Separation device and separation method

Abstract

A separation device, comprising a separation chip, a vacuum system and a frequency conversion module. The separation chip comprises a sample pool, two first chambers and two second chambers, each first chamber is connected to the sample pool through a first filter membrane, each first chamber is connected to the second chamber adjacent thereto through a second filter membrane, and the pore size of the second filter membrane is smaller than the pore size of the first filter membrane; two first vibrators are respectively arranged on the outer surfaces of the two second chambers; the frequency conversion module is respectively connected to the two second chambers through the vacuum system, and controls the vacuum system to alternately generate negative pressure in the chambers on both sides of the sample pool to achieve separation of liquid samples. The present application also provides a separation method. The separation device of the present application has a simple structure, can simultaneously separate target particles of multiple characteristic sizes, and has high separation efficiency.

Description

Technical Field

[0001] The present application relates to the technical field of extracellular vesicle separation, and in particular, to a separation device and a separation method for separating target particles of multiple characteristic sizes in a liquid sample. Background Art

[0002] Extracellular vesicles (EVs) are small biphospholipid membrane vesicles that are continuously secreted in large quantities by living cells. As carriers of intercellular communication, they carry specific components such as proteins, nucleic acids, and metabolic small molecules derived from mother cells. A large number of studies have shown that extracellular vesicles are involved in processes such as cell communication, cell migration, angiogenesis, and tumor cell growth. They are widely present in various body fluids and cell supernatants, and stably carry some important signaling molecules. The study of the functions related to extracellular vesicles has become a research hotspot and is expected to play a role in the early diagnosis of a variety of diseases.

[0003] At present, the main obstacle to the clinical application of extracellular vesicles is how to separate extracellular vesicles and their subpopulations from complex biological fluids. The traditional separation process is cumbersome and can only separate one target particle at a time, which takes a long time, has low separation efficiency, low throughput and low purity. Summary of the invention

[0004] In view of this, it is necessary to provide a separation device and a separation method that can solve the above problems.

[0005] The present application provides a separation device for separating and purifying a plurality of target particles from a liquid sample. The separation device comprises:

[0006] A separation chip, comprising a sample pool, a first chamber located at a first side of the sample pool, a first chamber located at a second side of the sample pool, and a second chamber located at a side of each of the first chambers away from the sample pool, each of the first chambers being connected to the sample pool via a first filter membrane, each of the first chambers being connected to the second chamber adjacent thereto via a second filter membrane, and the pore size of the second filter membrane being smaller than the pore size of the first filter membrane;

[0007] Vacuum system; and

[0008] A frequency conversion module is connected to the two second chambers respectively through the vacuum system, and the frequency conversion module is used to control the vacuum system to alternately generate negative pressure in the first chamber and the second chamber located on the first side and the first chamber and the second chamber located on the second side.

[0009] In some possible embodiments, the separation device further includes two first vibrators, one of which is located outside a corresponding second chamber; when the vacuum system stops generating negative pressure in the first chamber and the second chamber located on the first side and in the first chamber and the second chamber located on the second side, the two first vibrators are used to generate a first vibration wave.

[0010] In some possible implementations, the separation device further includes two second vibrating members, each of which is disposed on one side of a corresponding second filter membrane, and the second vibrating member is used to generate a second vibration wave.

[0011] In some possible implementations, the vibration frequency of the first vibration wave is lower than the vibration frequency of the second vibration wave.

[0012] In some possible embodiments, the separation chip also includes a chip body, which includes a first shell, a second shell, a third shell and a fourth shell combined in sequence; two second filter membranes are respectively arranged on the first shell and the fourth shell, and two second chambers are respectively arranged on the first shell and the fourth shell; two second filter membranes are respectively arranged on the second shell and the third shell, the first shell and the second shell together form one first chamber, and the third shell and the fourth shell together form another second chamber; the second shell and the third shell together form a sample pool.

[0013] In some possible embodiments, the two first vibration members are respectively attached to the outer surfaces of the first shell and the fourth shell, the two second vibration members are respectively fixed on the inner walls of the first shell and the second shell, and each second vibration member is arranged at a distance from a corresponding second filter membrane.

[0014] In some possible embodiments, the first shell and the fourth shell each include a first opening, each of the first openings is connected to the vacuum system, the chip body also includes a detachable cover, the cover is provided with a second opening corresponding to the sample pool, and the inner top wall of the cover is provided with a sealing gasket corresponding to the first shell, the second shell, the third shell and the fourth shell.

[0015] The present application also provides a separation method for separating and purifying target particles of various characteristic sizes from a liquid sample, the method comprising:

[0016] Providing the separation device as described above, and providing a liquid sample to the sample pool;

[0017] A negative pressure is generated in the first chamber and the second chamber located at the first side of the sample pool, so that components in the liquid sample in the sample pool whose particle size is smaller than the pore size of the first filter membrane enter the first chamber located at the first side under the action of the negative pressure, and components whose particle size is smaller than the pore size of the second filter membrane enter the second chamber located at the first side under the action of the negative pressure;

[0018] Stop generating negative pressure in the first chamber and the second chamber located on the first side, and generate negative pressure in the first chamber and the second chamber located on the second side of the sample pool, so that components in the liquid sample in the sample pool with a particle size smaller than the pore size of the first filter membrane enter the first chamber located on the second side under the action of negative pressure, and components with a particle size smaller than the pore size of the second filter membrane enter the second chamber located on the second side under the action of negative pressure; and

[0019] The generation of negative pressure in the first chamber and the second chamber located on the second side is stopped.

[0020] In some possible implementations, after stopping generating negative pressure in the first chamber and the second chamber located on the first side and stopping generating negative pressure in the first chamber and the second chamber located on the second side, the separation method further includes:

[0021] A first vibration wave is provided to the separation chip.

[0022] In some possible implementations, after stopping generating negative pressure in the first chamber and the second chamber located on the first side and stopping generating negative pressure in the first chamber and the second chamber located on the second side, the separation method further includes:

[0023] A second vibration wave is provided to the second filter membrane, wherein a frequency of the first vibration wave is lower than a frequency of the second vibration wave.

[0024] Compared with the prior art, the present application provides multiple chambers on both sides of the sample pool, and the multiple chambers are isolated by filter membranes with different pore sizes, so as to achieve the separation and purification of target particles of various characteristic sizes, with high purification efficiency and good purification effect; the separation device has a simple structure, and multiple independently designed shells are simple to form and easy to assemble. At the same time, the number of shells can be set according to actual needs, and the separation chambers can be increased or decreased, thereby improving the application scope of the separation device; the joint use of multiple groups of vibrating parts can enhance the anti-blocking effect and further improve the separation effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A schematic diagram of the functional modules of a separation device provided in one embodiment of the present application.

[0026] Figure 2 A schematic cross-sectional structure diagram of a separation chip provided in one embodiment of the present application.

[0027] Figure 3 An exploded view of a separation chip provided in an embodiment of the present application.

[0028] Figure 4 Schematic diagram of the structure of the first filter membrane in one embodiment of the present application.

[0029] Figure 5 It is a schematic structural diagram of the first shell and the first filter membrane in one embodiment of the present application.

[0030] Figure 6 for Figure 3 Cross-sectional view of the middle cover along VI-VI.

[0031] Figure 7 This is a schematic cross-sectional structure diagram of a separation chip provided in another embodiment of the present application.

[0032] Figure 8 This is an assembly diagram of a separation chip provided in another embodiment of the present application.

[0033] Fig. 9 An exploded view of a separation chip provided in another embodiment of the present application.

[0034] Fig.10 A schematic cross-sectional structure diagram of a separation chip provided in yet another embodiment of the present application.

[0035] Fig.11 Schematic diagram of the separation chip provided in an embodiment of the present application for separating a liquid sample.

[0036] Fig.12 A schematic diagram of a separation chip for separating a liquid sample according to another embodiment of the present application.

[0037] Fig.13 This is a schematic diagram of the functional modules of a separation device provided in another embodiment of the present application.

[0038] Main component symbols

[0039]

[0040]

[0041]

[0042] The following specific implementation methods will further illustrate the present application in conjunction with the above-mentioned drawings. DETAILED DESCRIPTION

[0043] The technical solution of the present application will be described below in conjunction with the preferred implementation modes and embodiments of the present application. It should be noted that when a unit is described as being "connected" to another unit, it may be directly connected to the other unit or there may be a central unit at the same time. When a unit is described as being "disposed on" another unit, it may be directly disposed on the other unit or there may be a central unit at the same time. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by technicians in the technical field of the present application. The names of the elements or devices used in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application.

[0044] See also Figure 1 , an embodiment of the present application provides a separation device 100, which is used to perform multi-stage separation and purification of particles of different sizes in a liquid sample to obtain target particles of multiple specific sizes. The liquid sample can be human plasma, serum, cerebrospinal fluid, saliva, urine, and gastric juice. The target particles can be exosomes or circulating tumor cells. The separation device 100 includes a separation chip 10, a vacuum system 30 connected to the separation chip 10, a frequency conversion module 40, and a controller 50 for controlling the frequency conversion module 40.

[0045] See also Figure 1 and Figure 2 The separation chip 10 includes a sample pool 12, a first chamber 13 located on a first side a of the sample pool 12, and a second chamber 14 adjacent to the first chamber 13 and away from the sample pool 12, and a first chamber 13 located on a second side b of the sample pool 12, and a second chamber 14 adjacent to the first chamber 13 and away from the sample pool 12, wherein the two first chambers 13 are symmetrically arranged, and the two second chambers 14 are symmetrically arranged. Each first chamber 13 is away from the second chamber 14 on one side of the sample pool 12. The sample pool 12 is used to accommodate a liquid sample. Each first chamber 13 is connected to the sample pool 12 through a first filter membrane 15, and each first chamber 13 is connected to the second chamber 14 adjacent thereto through a second filter membrane 16. The pore size of the second filter membrane 16 is smaller than the pore size of the first filter membrane 15. The frequency conversion module 40 is connected to the two second chambers 14 through the vacuum system 30 , respectively. The frequency conversion module 40 is used to control the vacuum system 30 to alternately generate negative pressure in the two first chambers 13 and the two second chambers 14 under the control of the controller 50 .

[0046] See also Figures 1 to 3The separation chip 10 further includes a chip body 11, which includes a first shell 111, a second shell 112, a third shell 113 and a fourth shell 114, which are sequentially combined. A second chamber 14 is provided on the first shell 111 and the fourth shell 114 respectively. The first shell 111 and the second shell 112 together form a first chamber 13, the third shell 113 and the fourth shell 114 together form another second chamber 14, and the second shell 112 and the third shell 113 together form a sample pool 12.

[0047] The first housing 111 includes a first receiving groove 1111 opening toward the second housing 112, a second filter membrane 16 is fixed at the opening of the first receiving groove 1111, and the first receiving groove 1111 and the second filter membrane 16 enclose a second chamber 14. The fourth housing 114 includes a second receiving groove (not shown) opening toward the third housing 113, the structure of the second receiving groove is substantially the same as that of the first receiving groove 1111, another second filter membrane 16 is fixed at the opening of the second receiving groove, and the second receiving groove and the second filter membrane 16 fixed thereon together enclose another second chamber 14. The second housing 112 includes a side wall 1121 and a through hole 1122 penetrating the side wall 1121, a first filter membrane 15 is fixed on the side wall 1121, and the first filter membrane 15 covers the through hole 1122, and the first housing 111 and the second housing 112 are assembled to enclose a first chamber 13. The third shell 113 includes a side wall 1131 and a through hole 1132 running through the side wall 1131, another first filter membrane 15 is fixed on the side wall 1131, and the first filter membrane 15 covers the through hole 1132. The third shell 113 and the fourth shell 114 are assembled to form another first chamber 13. The second shell 112 and the third shell 113 are assembled to form a sample pool 12. The separation chip 10 is formed by assembling four independent shells, which has a simple structure, is easy to shape, and is easy to assemble. It can be understood that the number of shells can be increased or decreased according to actual needs to form different numbers of chambers, thereby separating target particles of more sizes.

[0048] The chip body 11 of the separation chip 10 can be made of plastic, glass, metal or composite material. In one embodiment, the chip body 11 can be made of polymethyl methacrylate (PMMA) material by CNC or injection molding, and the four shells of the plastic material can be connected together by hot pressing bonding or fixed together by adhesive layer to achieve the purpose of assembly and sealing.

[0049] It is understandable that the volume of the sample pool 12 can be designed according to the actual application scenario. For the application scenario of biological biopsy, the volume of the sample pool 12 can be between 0.1-10 ml, and further between 0.6-2.5 ml.

[0050] See also Figure 1 and Figure 2 The sample pool 12 is provided with a sample adding port 121, which connects the sample pool 12 with the outside world. Liquid sample is added into the sample pool 12 through the sample adding port 121, and the separated target particles of a specific size are taken out through the sample adding port 121. The two second chambers 14 are both provided with a first opening 141. Specifically, the two first openings 141 are respectively opened on the side walls of the first shell 111 and the fourth shell 114, and the two first openings 141 respectively connect the two second chambers 14 with the outside world. The two first chambers 13 are both provided with a third opening 131, which connects the first chamber 13 with the outside world, and the separated target particles of another specific size are taken out through the third opening 131. It can be understood that when the separation chip 10 is working, the two third openings 131 are in a sealed state, and only the sample loading port 121 remains connected to the outside world. In this way, after the two first openings 141 are connected by the vacuum system 30 and alternately sucked, it can be ensured that the liquid sample in the sample pool 12 can only move unilaterally to the first side a or the second side b, thereby ensuring that the separated target particles will not be sucked back into the sample pool 12 in the opposite direction.

[0051] See also Figures 2 to 5 , the pore size of the first filter membrane 15 is larger than the pore size of the second filter membrane 16. During the separation process, the separation chip 10 can filter out two types of target particles of specific sizes, and the two first filter membranes 15 and the two second filter membranes 16 can filter out two types of target particles of different sizes. In this embodiment, the pore sizes of the two first filter membranes 15 are in the same range; the pore sizes of the two second filter membranes 16 are in the same range. The separated liquid sample mainly contains two types of extracellular vesicles (EVs), one is small extracellular vesicles (SEVs, with a size in the range of 30-200nm), and the other is large extracellular vesicles (LEVs, with a size in the range of 200-1000nm). In addition to the two types of EVs, the liquid sample also contains relatively small free pollutants (such as free proteins and nucleic acids). By filtering through the first filter membrane 15, LEV can be separated from other components, and LEV is retained in the sample pool 12. Further by filtering through the second filter membrane 16, SEV can be separated from contaminants, and SEV is retained in the two first chambers 13. The contaminants are separated into the second chamber 14 and then discharged from the separation chip 10 through the two first openings 141.

[0052] The two first filter membranes 15 and the two second filter membranes 16 can be made of the same membrane material or different membrane materials. Each first filter membrane 15 or each second filter membrane 16 can be made of one membrane material or a composite of multiple membrane materials. The first filter membrane 15 and the second filter membrane 16 can be porous materials, including but not limited to porous ceramic materials, porous plastic materials and porous metal materials. Specifically, the first filter membrane 15 and the second filter membrane 16 can be selected from one or more of anodized aluminum membrane, cellulose acetate membrane, polyethylene membrane, polypropylene membrane and polystyrene membrane. In the present embodiment, the first filter membrane 15 and the second filter membrane 16 are porous ceramic membranes, such as anodized aluminum membranes.

[0053] In this embodiment, the first filter membrane 15 includes a filter membrane body 151, a membrane hole 152 provided on the filter membrane body 151, and a connection layer 153 provided on the edge of the filter membrane body, and the connection with the second shell 112 and the third shell 113 is achieved through the connection layer 153. The connection layer 153 can be a plastic film attached to the filter membrane body 151, and the plastic film is connected to the shell of plastic material by hot pressing bonding. It can be understood that the connection layer 153 can also be a glue layer, and the first filter membrane 15 is directly connected to the shell through the glue layer.

[0054] It can be understood that the structure of the second filter membrane 16 is similar to that of the first filter membrane 15, and the specific connection method with the housing can be found in the above description.

[0055] It is understandable that when more chambers need to be formed to separate more target particles of a specific size, a filter membrane can be further provided, for example, a third filter membrane (not shown) is provided on the side of the second filter membrane 16 away from the first filter membrane 15, and the pore size of the third filter membrane is smaller than that of the second filter membrane 16, so as to separate components smaller than SEV. It is also understandable that a fourth filter membrane (not shown) can be added between the first filter membrane 15 and the second filter membrane 16, and the pore size of the fourth filter membrane is between that of the first filter membrane 15 and the second filter membrane 16, so as to separate target particles with a size between SEV and LEV.

[0056] In one embodiment, if Figure 2 and Figure 6As shown, the separation chip 10 also includes a cover plate 17, which is used to cover the chip body 11 to seal the two third openings 131. The cover plate 17 includes a cover plate body 171, a second opening 172, a positioning plate 173 provided on both sides of the second opening 172 and located on the inner top wall of the cover plate body 171, and a sealing gasket 174 provided on the inner top wall of the cover plate body 171. The second opening 172 is connected to the sample loading port 121. When the cover plate 17 is covered on the chip body 11, the positioning plate 173 is clamped on the side wall of the second shell 112 and the third shell 113 close to the sample pool 12 to position the cover plate 17 and the chip body 11, and the cover plate 17 clamps the chip body 11. The sealing gasket 174 is located between the inner top wall of the cover plate body 171 and each shell to achieve the purpose of sealing. The sealing gasket 174 can be a rubber pad or a glue layer.

[0057] In one embodiment, if Figure 2 As shown, the outer surfaces of the first shell 111 and the fourth shell 114 are provided with connecting pipes 115 , and each connecting pipe 115 is connected to a corresponding first opening 141 to achieve communication with the vacuum system 30 .

[0058] like Figures 1 to 5 As shown, combined Fig.11 and Fig.12When the separation chip 10 is used, a liquid sample is added to the sample pool 12, and the two first openings 141 are respectively connected to the vacuum system 30. When the vacuum system 30 sucks the first chamber 13 and the second chamber 14 located at the first side a through the first opening 141 located at the first side a of the sample pool 12, negative pressure is generated in the first chamber 13 and the second chamber 14 at the first side a. Under the action of negative pressure in the first chamber 13 and the second chamber 14 of the first side a, the components (including SEV, small particle pollutants and liquid components) in the liquid sample in the sample pool 12 whose size is smaller than the pore size of the first filter membrane 15 move toward the first filter membrane 15 of the first side a, and flow into the first chamber 13 of the first side a through the first filter membrane 15 of the first side a, thereby leaving the components whose size is larger than the pore size of the first filter membrane 15 in the sample pool 12; the SEV, small particle pollutants and liquid components entering the first chamber 13 of the first side a further pass through the second filter membrane 16 of the first side a, and the components (including small particle pollutants and liquid components) whose size is smaller than the pore size of the second filter membrane 16 pass through the second filter membrane 16 and enter the second chamber 14 of the first side a, and finally exit the separation chip 10 through the first opening 141 of the first side a. When the vacuum system 30 sucks the first chamber 13 and the second chamber 14 located on the second side b through the first opening 141 located on the second side b of the sample pool 12, negative pressure is generated in the first chamber 13 and the second chamber 14 of the second side b. Under the action of negative pressure, the two different sizes of target particles in the liquid sample in the sample pool 12 are respectively retained in the sample pool 12 and the first chamber 13 on the second side b, and the small particle pollutants and the liquid components enter the second chamber 14. At the same time, the liquid sample in the sample pool 12 will produce a backflow phenomenon at the first filter membrane 15 and the second filter membrane 16 on the second side b, thereby reducing or removing the components adhering to the first filter membrane 15 and the second filter membrane 16 on the second side b, and avoiding the occurrence of filter membrane blockage during the filtration and separation process. Repeatedly and alternately generating negative pressure in the chambers located on the first side a and the second side b of the sample pool 12 can effectively make the liquid sample repeatedly and alternately flow through the filter membranes located on the first side a and the second side b of the sample pool 12, so that the two different sizes of components in the liquid sample (i.e., the two target particles) are respectively retained in the sample pool 12 and the two first chambers 13. The structural design of the separation chip 10 can separate two components of different sizes in the liquid sample at the same time, thereby improving the separation efficiency; the separation process of the two target particles is completed automatically, which can eliminate the deviation caused by operation; moreover, the vacuum system 30 operates alternately, so that the components adsorbed on the surface of the first filter membrane 15 and the second filter membrane 16 are easy to fall off from the surface of the filter membrane in the repeated alternating negative pressure changes, which can effectively prevent the membrane pores of the filter membrane from being blocked.

[0059] Please refer to Figure 1 and Figure 2In one embodiment, the vacuum system 30 alternately generates negative pressure in the first chamber 13 and the second chamber 14 located at the first side a and the first chamber 13 and the second chamber 14 located at the second side b to form a periodic trapezoidal pulse signal. The trapezoidal pulse signal can prevent the two first filter membranes 15 and the two second filter membranes 16 from being damaged by a sudden change in the direction of the negative pressure. In one embodiment, in view of the high protein content in the plasma sample, in order to further avoid the clogging of the filter membrane, negative pressure can be generated in the chamber on one side of the sample pool 12 while positive pressure can be generated in the chamber on the other side of the sample pool 12 to strengthen the reflux phenomenon at the filter membrane.

[0060] See also Figures 7 to 9 The separation device 100 further includes a first vibrating member 20 disposed outside each of the second chambers 14. The controller 50 is further used to control each of the first vibrating members 20 to vibrate to generate a first vibration wave when the vacuum system 30 stops generating negative pressure in the two first chambers 13 and the two second chambers 14. The number of the first vibrating members 20 is two. One of the first vibrating members 20 is disposed on the outer surface of the second chamber 14 on the first side a (i.e., the outer surface of the first shell 111). The other first vibrating member 20 is disposed on the outer surface of the second chamber 14 on the second side b (i.e., the outer surface of the fourth shell 114). The first vibrating member 20 is used to generate a transverse first vibration wave when vibrating, and then transmit the first vibration wave to the first shell 111 and the fourth shell 114. The second vibration wave is transmitted to the entire separation chip 10 through the first shell 111 and the fourth shell 114, so that the separation chip 10 vibrates at a low frequency, so that the target particles adsorbed in the membrane pores of the filter membrane are separated from the membrane pores of the filter membrane and resuspended in the refluxed liquid sample, thereby preventing the membrane pores of the filter membrane from being blocked.

[0061] In this embodiment, the two first vibrators 20 can be directly fixed on the first shell 111 and the fourth shell 114. Alternatively, the first vibrators 20 can be independent. When the separation chip is used, the two first vibrators 20 are respectively attached to the outer surfaces of the first shell 111 and the fourth shell 114, so as to provide vibration waves to the separation chip 10. The independent arrangement of the first vibrators 20 can reduce the cost of the separation chip 10.

[0062] In this embodiment, the first vibrating element 20 may be a low-frequency harmonic oscillator or a vibration motor.

[0063] like Fig.10As shown, the two second filter membranes 16 are respectively provided with second vibrators 60, and the number of the second vibrators 60 is two. One of the second vibrators 60 is arranged at a distance from the second filter membrane 16 on the first side a. The other second vibrator 60 is arranged at a distance from the second filter membrane 16 on the second side b. The second vibrator 60 is used to generate a transverse second vibration wave when vibrating, and then transmit the second vibration wave to the two second filter membranes 16, so as to achieve high-frequency resonance with the two second filter membranes 16. Among them, the frequency of the second vibration wave is greater than the frequency of the first vibration wave. Therefore, the target particles adsorbed in the membrane pores of the first filter membrane 15 are separated from the membrane pores of the filter membrane and resuspended in the refluxed liquid sample, thereby further preventing the membrane pores of the first filter membrane 15 from being blocked and achieving efficient separation. The second vibration wave can disturb the liquid sample and the filter membrane together with the first vibration wave and generate an acoustic flow effect, thereby preventing the target particles from blocking the filter pores or agglomerating together, and improving the separation and purification efficiency. In one embodiment, the second vibrator 60 can be a high-frequency harmonic oscillator.

[0064] In this embodiment, the second vibrating member 60 is directly fixed on the inner walls of the first shell 111 and the fourth shell 114. A certain distance is maintained between the second vibrating member 60 and the second filter membrane 16. The distance h is in the range of 0.3 to 0.6 mm, and specifically can be 0.5 mm. This distance enables the second vibrating member 60 to ensure resonance with the second filter membrane 16 while ensuring that the separated particles can pass through the second filter membrane 16 smoothly and will not clog the membrane pores of the second filter membrane 16.

[0065] It is understandable that a third vibrator (not shown) can also be provided on the two first filter membranes 15, and the number of the third vibrators is two. One of the third vibrators is fixed to the surface of the first filter membrane 15 on the first side a. The other third vibrator is fixed to the surface of the first filter membrane 15 on the second side b. The third vibrator is used to generate a transverse third vibration wave when vibrating, and then transmit the third vibration wave to the two first filter membranes 15, thereby driving the two first filter membranes 15 to vibrate at a low frequency, and the frequency of the third vibration wave is less than the frequency of the second vibration wave. The third vibration wave combined with the second vibration wave can disturb the liquid sample and the filter membrane together with the first vibration wave and produce an acoustic flow effect, further preventing the target particles from clogging the filter holes or agglomerating together, and further improving the separation and purification efficiency. In one embodiment, the third vibrator can be a low-frequency harmonic oscillator or a vibration motor. It is worth noting that in this embodiment, since the aperture of the first filter membrane 15 is large, it is not easy to be blocked, and there is no need to use high-frequency vibration to prevent blockage. Therefore, the third vibrator is not provided on the first filter membrane 15.

[0066] In one embodiment, the first vibrator 20, the second vibrator 60 and the third vibrator can be located on the same horizontal plane and coaxially arranged, so that the first vibration wave, the second vibration wave and the third vibration wave can be on the same horizontal plane and can be superimposed on each other to form a coordinated vibration.

[0067] See also Figures 1 to 3 , Figures 7 to 10 , the vacuum system 30 is used to respectively generate negative pressure in the two groups of chambers of the separation chip 10 located on both sides of the sample pool 12. The vacuum system 30 can be two independent vacuum systems, or it can be a designed vacuum system. The vacuum system 30 can also include equipment such as a miniature vacuum pump or a miniature air pump. It is understandable that the vacuum system 30 and the separation chip 10 can be connected by a pipe with better air tightness. 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 141 on the first shell 111, and the second vacuum pump 320 is connected to the first opening 141 on the fourth shell 114.

[0068] The frequency conversion module 40 is electrically connected to 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 pressure is alternately generated in the chambers on both sides of the sample pool 12. In one embodiment, the frequency conversion module 40 includes a frequency converter 410 and a control valve 420 connected to the frequency converter 410. The control valve 420 can be a liquid circuit converter, including but not limited to a solenoid valve and a rotary valve. The control valve 420 is respectively connected to one of the first vacuum pump 310 and the second vacuum pump 320, so that the first vacuum pump 310 and the second vacuum pump 320 repeatedly work alternately. For example, the control valve 420 is connected to the first vacuum pump 310, so that the frequency converter 410 controls the first vacuum pump 310 to operate, and air is pumped through the first opening 141 on the first shell 111 to generate negative pressure in the first chamber 13 and the second chamber 14 on the first side a, so that the two target particles in the liquid sample in the sample pool 12 are separated, as described in the above process for details; then, the frequency converter 410 controls the first vacuum pump 310 to stop operating; thereafter, the control valve 420 is switched to be connected to the second vacuum pump 320, so that the frequency converter 410 controls the second vacuum pump 320 to operate, and air is pumped through the first opening 141 on the fourth shell 114 to generate negative pressure in the first chamber 13 and the second chamber 14 on the second side b, so that the two target particles in the liquid sample in the sample pool 12 are separated, as described in the above process for details; thereafter, the frequency converter 410 controls the second vacuum pump 320 to stop operating; and the above steps are repeated multiple times.

[0069] The controller 50 is electrically connected to the first vibrator 20 and the second vibrator 60. The controller 50 is used to control the first vibrator 20, the second vibrator 60 and the third vibrator 70 to vibrate when the first chamber 13 and the second chamber 14 located on the first side a stop suctioning, thereby generating the first vibration wave, the second vibration wave and the third vibration wave respectively. The controller 50 is also used to control the first vibrator 20, the second vibrator 60 and the third vibrator 70 to vibrate when the first chamber 13 and the second chamber 14 located on the second side b stop suctioning, thereby generating the first vibration wave, the second vibration wave and the third vibration wave respectively. Among them, the controller 50 can be electrically connected to the first vacuum pump 310 and the second vacuum pump 320. When the first vacuum pump 310 or the second vacuum pump 320 stops running, the controller 50 determines that the first vacuum pump 310 stops suctioning, or determines that the second vacuum pump 320 stops suctioning, and at this time, the corresponding first vibrator 20, the second vibrator 60 and the third vibrator 70 can be notified to start vibrating. The controller 50 may also be electrically connected to the frequency conversion module 40 to control the operation of the frequency conversion module 40 .

[0070] Combined with reference Figures 11 to 13 , combined with Figure 1 and Figure 2 The present embodiment further provides a method for separating a plurality of target particles in a liquid sample using the separation device 100, which comprises the following steps:

[0071] Step S1 : providing the separation device 100 of the present application, and adding a liquid sample into the sample pool 12 .

[0072] Step S2 : the vacuum system 30 sucks the first chamber 13 and the second chamber 14 located at the first side a through the first opening 141 located on the first shell 111 , so that negative pressure is generated in the first chamber 13 and the second chamber 14 located at the first side a.

[0073] In this way, the components in the liquid sample in the sample pool 12 whose sizes are smaller than the pore size of the first filter membrane 15 (including SEV, small particle pollutants and liquid components) move toward the first filter membrane 15 on the first side a, and flow into the first chamber 13 on the first side a through the first filter membrane 15 on the first side a, thereby leaving the components whose sizes are larger than the pore size of the first filter membrane 15 in the sample pool 12; the SEV, small particle pollutants and liquid components entering the first chamber 13 on the first side a further pass through the second filter membrane 16 on the first side a, and the components whose sizes are smaller than the pore size of the second filter membrane 16 (including small particle pollutants and liquid components) pass through the second filter membrane 16 into the second chamber 14 on the first side a, and finally are discharged from the separation chip 10 through the first opening 141 on the first side a.

[0074] Step S3, the vacuum system 30 stops sucking the first chamber 13 and the second chamber 14 located at the first side a, and controls the first vibrator 20 and the second vibrator 60 to vibrate, thereby generating the first vibration wave and the second vibration wave respectively. At the same time, the vacuum system 30 sucks the first chamber 13 and the second chamber 14 located at the second side b through the first opening 141 located on the fourth shell 114, so that negative pressure is generated in the first chamber 13 and the second chamber 14 located at the second side b.

[0075] The first vibration wave drives the entire separation chip 10 to vibrate at a low frequency to prevent particles from gathering together. The second vibration wave drives the second filter membrane 16 to vibrate at a high frequency, so that the target particles adsorbed in the membrane pores of the second filter membrane 16 can be quickly separated from the membrane pores of the second filter membrane 16 and resuspended in the refluxed liquid sample. At the same time, the vacuum system 30 sucks the first chamber 13 and the second chamber 14 located on the second side b through the first opening 141 located on the fourth shell 114, so that the first opening 141 located on the fourth shell 114 sucks the first chamber 13 and the second chamber 14 located on the second side b to generate negative pressure. The components adhered to the surface of the first filter membrane 15 and the second filter membrane 16 located on the first side a can be separated from the filter membrane surface along with the air flow and / or liquid flow and enter the suspended liquid. The two target particles of different sizes in the liquid sample in the sample pool 12 are respectively left in the sample pool 12 and the first chamber 13 on the second side b, and the small particle pollutants and the liquid components enter the second chamber 14.

[0076] Step S4 , the vacuum system 30 stops sucking the first opening 141 on the fourth shell 114 and sucks the first chamber 13 and the second chamber 14 on the second side b, and controls the first vibrator 20 and the second vibrator 60 to vibrate.

[0077] Then, steps S2 to S4 can be cycled multiple times, so that the pollutant components in the liquid sample that are smaller than the pore size of the filter membrane enter the second chamber 14 and are then removed, the SEV particles enter the first chamber 13, and the LEV particles are retained in the sample pool 12, so as to achieve better separation and purification of multiple target particles.

[0078] In summary, the present application arranges multiple chambers on both sides of the sample pool, and the multiple chambers are isolated by filter membranes with different pore sizes, so as to achieve the separation and purification of target particles of various characteristic sizes, with high purification efficiency and good purification effect; the separation device has a simple structure, and multiple independently designed shells are convenient for molding and assembly. At the same time, the number of shells can be set according to actual needs, and the separation chambers can be increased or decreased, thereby improving the application scope of the separation device 100; the joint use of multiple groups of vibrating parts can enhance the anti-blocking effect and further improve the separation effect.

Claims

1. A separation device for separating and purifying a plurality of target particles from a liquid sample, characterized in that: The separation device comprises: A separation chip, comprising a sample pool, a first chamber located at a first side of the sample pool, a first chamber located at a second side of the sample pool, and a second chamber located at a side of each of the first chambers away from the sample pool, each of the first chambers being connected to the sample pool via a first filter membrane, each of the first chambers being connected to the second chamber adjacent thereto via a second filter membrane, the pore size of the second filter membrane being smaller than the pore size of the first filter membrane, the separation chip further comprising a chip body, the chip body comprising a first shell, a second shell, a third shell and a fourth shell which are sequentially combined, two of the second filter membranes being respectively arranged on the first shell and the fourth shell, two of the second chambers being respectively arranged on the first shell and the fourth shell; two of the second filter membranes being respectively arranged on the second shell and the third shell, the first shell and the second shell together enclose one of the first chambers, the third shell and the fourth shell together enclose another of the second chambers; the second shell and the third shell together enclose a sample pool, the chip body further comprising a cover plate detachably arranged on the chip body, the cover plate being provided with a second opening corresponding to the sample pool; Vacuum system; and a frequency conversion module, which is respectively connected to the two second chambers through the vacuum system, and is used to control the vacuum system to alternately generate negative pressure in the first chamber and the second chamber located on the first side and the first chamber and the second chamber located on the second side, The separation device further comprises two first vibrators, one of the first vibrators is located outside a corresponding second chamber, and the two first vibrators are respectively attached to the outer surfaces of the first shell and the second shell; when the vacuum system stops generating negative pressure in the first chamber and the second chamber located on the first side and in the first chamber and the second chamber located on the second side, the two first vibrators are used to generate a first vibration wave; The separation device also includes two second vibrating members, one of which is arranged on one side of a corresponding second filter membrane, and the two second vibrating members are respectively fixed on the inner walls of the first shell and the fourth shell, and each second vibrating member is arranged at a distance from a corresponding second filter membrane, and the second vibrating member is used to generate a second vibration wave.

2. The separation device according to claim 1, characterized in that The vibration frequency of the first vibration wave is lower than the vibration frequency of the second vibration wave.

3. The separation device according to claim 1, characterized in that The first shell and the fourth shell each include a first opening, and each of the first openings is connected to the vacuum system; The inner top wall of the cover plate is provided with sealing gaskets corresponding to the first shell, the second shell, the third shell and the fourth shell.

4. A separation method for separating and purifying target particles of various characteristic sizes from a liquid sample, characterized in that: The method comprises: Providing the separation device as claimed in claim 1, and providing a liquid sample to the sample pool; A negative pressure is generated in the first chamber and the second chamber located at the first side of the sample pool, so that components in the liquid sample in the sample pool whose particle size is smaller than the pore size of the first filter membrane enter the first chamber located at the first side under the action of the negative pressure, and components whose particle size is smaller than the pore size of the second filter membrane enter the second chamber located at the first side under the action of the negative pressure; Stop generating negative pressure in the first chamber and the second chamber located on the first side, and generate negative pressure in the first chamber and the second chamber located on the second side of the sample pool, so that components in the liquid sample in the sample pool with a particle size smaller than the pore size of the first filter membrane enter the first chamber located on the second side under the action of negative pressure, and components with a particle size smaller than the pore size of the second filter membrane enter the second chamber located on the second side under the action of negative pressure; and The generation of negative pressure in the first chamber and the second chamber located on the second side is stopped.

5. The separation method according to claim 4, characterized in that After the stopping of generating the negative pressure in the first chamber and the second chamber located at the first side and the stopping of generating the negative pressure in the first chamber and the second chamber located at the second side, the separation method further includes: A first vibration wave is provided to the separation chip.

6. The separation method according to claim 5, characterized in that After the stopping of generating the negative pressure in the first chamber and the second chamber located at the first side and the stopping of generating the negative pressure in the first chamber and the second chamber located at the second side, the separation method further includes: A second vibration wave is provided to the second filter membrane, wherein a frequency of the first vibration wave is lower than a frequency of the second vibration wave.

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