Separation system of target substances in biological fluid samples

Abstract

The present invention provides a separation system for target substances in biological liquid samples, the system comprising: a microporous array membrane, the microporous array membrane is used to receive or contact the biological liquid sample; a vibration power source element capable of vibrating, and a transmission element that transmits the vibration emitted by the vibration power source to the microporous array membrane. By using this system, not only can the target substances in the biological liquid be separated efficiently through the membrane, but also the biological activity of the target substances can be retained to the maximum extent without being damaged.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular, to a system and method for separating special cells in a sample by using a microporous array filter membrane. Background Art

[0002] High-porosity microporous array filters are suitable for the concentration and separation of clinical or biological fluid samples. Although microporous array filters usually have higher porosity and lower membrane resistance than traditional chemical membranes, generally, pore clogging is not likely to occur during the separation of appropriate amounts of clinical and biological fluid samples.

[0003] However, when the number of cells or particles in the liquid sample far exceeds the number of micropores, congestion will inevitably occur, and even complete pore blockage will occur, making it impossible to complete the filtration. In actual applications, the number of white blood cells in 1mL of normal human blood is 4 million to 10 million, and the number of red blood cells is hundreds of millions. If the number of micropores in the working area is far lower than the number of cells, especially when the number of target cells intercepted on the membrane exceeds a certain number, a certain degree of congestion will occur. When congestion occurs, not only will the time for cell separation increase exponentially, but the changes in the microflow field caused by the accumulation of "membrane scale" will also affect the separation accuracy and screening size. The membrane scale here can be understood as the accumulation of other non-target substances, such as white blood cells or red blood cells. Therefore, in practical applications, a solution that can effectively increase the separation flux and alleviate congestion is needed. Summary of the invention

[0004] In order to improve the separation effect of a high-throughput microporous array membrane on target substances in biological samples, the present invention provides a separation method and system, which can effectively solve some defects of traditional technologies.

[0005] On the one hand, the present invention provides a separation system, which includes: a microporous array membrane, which is used to receive or contact a biological sample; a vibration power source element capable of vibrating; and a transmission element that transmits the vibration emitted by the vibration power source to the microporous array membrane.

[0006] In some embodiments, the microporous array membrane includes 500-50,000,000 micropores / square millimeter per unit area, the diameter of the micropores is 0.1-50 microns, the thickness of the membrane is 1-100 microns, and the porosity is 5%-90%. From the processing type, it mainly refers to micro-processed membranes with precise holes, and also includes some chemical membranes and nuclear pore membranes with narrow size distribution and small membrane resistance. The membrane material can be polycarbonate (PC), polyimide (PI), polyparaxylene (Parylene), etc. The present invention preferably uses polyparaxylene microporous array membranes. The microporous structure can be circular, square, rectangular, regular hexagonal, etc. The following micropore diameter refers to the equivalent diameter corresponding to the inscribed circle of the micropore unless otherwise specified.

[0007] In some embodiments, the vibration driving power source includes various mechanical vibration generators such as hollow cup vibration motors and / or eccentric wheel DC motors; ultrasonic generators based on transducer chips and / or pneumatic vibration devices, etc.

[0008] In some embodiments, the transmission element includes elements such as spring transmission and metal rod transmission that can stably transmit vibration to the membrane. The transmission element includes a support plate, and the spring is arranged under the support plate to support the support plate, so that the support plate is supported by the spring to stand. In some embodiments, the spring is located under the support plate, and has three or more spring elements, so that the support plate is in a stable state, but is suspended by the spring. One end of the spring element is used to support the support plate, and the other end can be located on the operating table, so that the entire vibration system is in a relatively independent position.

[0009] In some embodiments, the membrane is disposed in a membrane carrier container, so that the carrier container includes a membrane and a cavity for receiving a sample, so that the liquid sample in the cavity is in direct contact with the membrane. In some embodiments, the container includes an ultra-low negative pressure element, which is located below the membrane and is used to apply a certain pressure to the membrane. There are many ways to apply pressure, such as a vacuum pump, or a cavity (lower cavity) with liquid in the cavity, and a certain pressure is applied by the gravity of the liquid. Of course, it is also possible to form a negative pressure below the membrane by means of the liquid sample passing through the membrane flowing into the cavity, thereby accelerating the liquid on the membrane (the liquid sample in the cavity for receiving the sample) to pass through the membrane, thereby allowing the sample to pass through the membrane more quickly, and allowing substances that cannot pass through the membrane, such as tumor cells, to stay on the membrane. At this time, the membrane is located between the cavity for receiving the sample and the lower cavity.

[0010] In some embodiments, the vibration driving power source is arranged on a support plate or fixedly connected to the support plate. When the vibration power source vibrates, the vibration can be transmitted to the support plate, and then transmitted to the membrane through the support plate. Thus, the target substance in the biological sample on the membrane is retained on the membrane, while the non-target substance in the sample flows out through the micropores of the membrane. In some embodiments, the vibration source includes one or two, and the power of the two vibration sources is different.

[0011] In some embodiments, the support plate includes a structure for mounting the membrane carrier container, through which the membrane carrier container is detachably fixed to the support plate. In this way, when the system is used, the membrane carrier container can be detachably replaced, so that the same system can be used to filter a variety of different biological samples with different membrane carrier containers. For example, each membrane carrier container filters a biological sample, and multiple membrane carrier containers can filter different biological samples multiple times. In some embodiments, the membrane carrier container includes an annular groove at the bottom, and a raised structure on the support plate, and the raised structure is snapped into the annular groove to play a fixing role. In some embodiments, a limit block is set on the support plate, and the limit block is snapped onto the container so that the position of the container does not change. In this way, in fact, the container and the support plate are connected as an integrated structure, which facilitates the transmission of the vibration waves emitted by the vibration source to the film of the container.

[0012] In some embodiments, the target substances in the biological sample are tumor cells, abnormal circulating cells, shed tumor cells, cell lines capable of producing antibodies or antigens, lymphocytes or stem cells of autologous or allogeneic origin, adipocytes, macrophages, fungal spores, bacteria, viruses, extracellular vesicles, or other substances with biological activity. These substances generally cannot pass through the micropores or submicron pores of the membrane, while other non-target substances in the sample can pass through the micropores, such as water molecules, cells / particles smaller than the micropores, and cells / particles with strong deformability, thereby achieving the purpose of filtration separation.

[0013] In some embodiments, the system also includes an ultra-low pressure separation system for pushing a liquid sample having various cells and other substances randomly distributed onto the surface of a microporous array membrane under a stable ultra-low pressure for screening based on the membrane surface structure and pore size.

[0014] Under the effect of the ultra-low pressure microporous array membrane separation strategy, as the separation time progresses, non-target substances and target substances are gradually enriched on both sides of the membrane, achieving a separation or concentration effect. However, as the filtration proceeds, according to the designed separation strategy, the accumulation of substances on both sides of the membrane improves the orderliness of the overall sample. When a large number of ordered cells or substances accumulate on one side of the membrane and the original randomly distributed liquid sample cannot effectively contact the membrane, it is generally believed that the sample on the membrane surface has a serious "polarization" phenomenon, and it is impossible to further efficiently separate and improve the orderliness of the sample, thereby affecting the separation effect and efficiency of the target substance ( Fig. 9). At this time, an efficient method is needed to depolarize and re-randomize the local sample. The characteristic of ultra-low pressure microporous array filter membrane is that the surface order is far greater than that of traditional chemical membranes, so the local polarization degree of the sample on the membrane surface is very high. However, the vibration of the present invention just overcomes the inherent defects or shortcomings of the ultra-low pressure microporous array membrane separation strategy alone. The efficient local mixing brought by vibration is a very effective depolarization solution. Therefore, if the vibration separation method of the present invention is added under ultra-low pressure, separation and / or concentration can be performed faster and better.

[0015] In some embodiments, the system further comprises a vibration control system for controlling the frequency of vibrations emitted by the vibration driven power source.

[0016] In the present invention, when the vibration wave of the rated frequency is emitted by the vibration driving power source, it is transmitted to the membrane through the transmission element (support plate or support plate with spring), so that the biological sample on the membrane also vibrates. This vibration causes different substances in the sample, such as different cells, to vibrate. This vibration causes the target substance, such as tumor cells, to detach from the surface of the membrane, thereby allowing non-target substances, such as water molecules and other cells to pass through the micropores, so that only the target substance is on the surface of the membrane, thereby achieving the purpose of separation. On the other hand, this vibration not only drives the target substance to detach from the surface of the membrane, but also drives the substance on the membrane to move in a lateral position. This lateral position movement is generally difficult to see with the naked eye. This tiny displacement is caused by the transmission element. When the vibration occurs, the support plate and the spring naturally move slightly left and right, thereby driving the substance on the membrane to move in position while detaching from the membrane surface. In this way, the flow and depolarization of non-target substances and target substances can be accelerated, the randomness of the sample on the membrane surface can be enhanced, and the high efficiency of the sample separation on the membrane surface can be maintained, so that the purpose of rapid separation of the target substance can be achieved. This is a possible technical principle of the present invention explained in principle. This can ensure both the efficiency of separation and the quality of the target substance to be separated, for example, cells will not be damaged ( Fig. 9 rightmost diagram of ).

[0017] This separation principle is different from the principle of depolarization optimization separation using the traditional vortex method. Fig. 9), in fact, the sample as a whole produces a vortex mixing effect by rotating centrifugal or eccentric motion. If the shear field generated by the vortex relative to the membrane surface is large enough, the cells can be separated from the membrane surface, thereby achieving the effect of sample depolarization. However, this separation method often does not consider the activity of the target substance, but removes impurities in the sample. This is because when the liquid is vortexed, it is necessary to generate a shear field that can affect the liquid film and cells on the membrane surface, especially the cells in the center of the membrane. Then, the shear field distribution on the membrane is enhanced along the center toward the inner wall end of the cavity (including the entire inner wall of the cavity), which often satisfies the sample depolarization in the center of the membrane, and will cause excessive shear force damage to the cells near the edge of the membrane and the inner wall of the cavity, damaging the biological activity of the sample, especially for the target cells staying on the membrane surface. Under high shear force, the cells are broken or damaged, which significantly affects the activity and re-culture survival rate of the target cells. In comparison, the vibration scheme of the present invention can form a uniform and stable sample depolarization effect on the membrane surface, which can more efficiently achieve the requirements of anti-blocking and improving separation efficiency without affecting separation accuracy and cell activity.

[0018] In addition, when the present invention separates the target substance, it is not desirable that the activity of the separated substance, such as a tumor cell or a cell line producing antigen and antibody, is affected, such as the cell stress signaling pathway is activated, apoptotic, or even ruptured. In this way, although the purpose of separation is achieved, if the separated cells are ruptured or the biological function is affected, they cannot be used subsequently. For example, after the separated tumor cells are collected, subsequent culture and other studies such as drug sensitivity are carried out. After the cells producing antigen and antibody are separated, they continue to be cultured to produce antibodies (cell perfusion). Therefore, the frequency of vibration should not be too high or too low. If it is too low, the separation effect cannot be achieved or the separation efficiency is low, and the time spent is long. If it is too high, it may cause damage to the target cells.

[0019] Microporous array membrane: The microporous array membrane of the present invention refers to a microporous array membrane or chip with precise pores or a narrow pore size distribution range (within 5%). The micropores can be round, square, hexagonal, long strip, or irregular. The micropore density is high (generally the porosity is greater than 20%), and the ratio of the membrane or chip thickness to the pore size does not exceed 10 times. Such microporous array membranes include chemical membranes with very precise micropore distribution (such as alumina separation membranes, polystyrene separation membranes, etc.), nuclear pore membranes with relatively uniform micropore distribution (polyethylene terephthalate microporous membranes, or polycarbonate microporous membranes, etc.), and more are micro-machined membranes prepared by micro-electromechanical processes (MEMS processes such as photolithography, etching, and imprinting), including polyimide (PI) microporous array membranes, polypropylene microporous array membranes, various types of photosensitive resin microporous array membranes, polyparaxylene microporous array membranes, etc. These membranes can be used by the invented vibration device to analyze the target substance in the liquid sample. In some specific embodiments, polycarbonate nuclear pore membrane, polyimide microporous array membrane, and polyparaxylene microporous array membrane are used as test objects. When the mechanical strength of the membrane material itself can withstand the vibration intensity, and the micropore pore size is not particularly close to the size of the target separation object to ensure that the material itself has little effect on the interception of the target separation object, the above-mentioned various types of membranes have the same effect trend on cell separation with the assistance of the vibration system of the present invention. There may be some changes in the separation performance curve due to the difference in porosity and membrane resistance, but the advantages of the present invention can be reflected in general. Among them, the polyparaxylene microporous array membrane has the best performance, and the separation with the assistance of the vibration system is stable, efficient, and has little cell loss. Among them, for the recovery and re-cultivation after cell separation, since some cells will be stuck in the membrane pores, the performance of the membrane surface and the adsorption of the membrane material itself to the cells will affect the recovery rate and re-cultivation activity. The hexagonal polyimide microporous array membrane and polyparaxylene microporous array membrane in the present invention have the best application effect on the recovery and re-cultivation. Under the same vibration conditions and the optimal separation membrane pore size, the separation efficiency is high, the recovery rate is high, and the cell damage is small.

[0020] The microporous membrane with the above characteristics has uniform and low resistance to membrane flow, and usually works at ultra-low pressure (separation pressure does not exceed 100KPa). Vibration of a certain frequency can very stably cause relative displacement between the membrane and the liquid sample, thereby forming a tangential shear flow, which can prevent blockage and increase filtration flux without affecting separation accuracy. This shear flow occurs in a very microscopic state, around each pore of the membrane, so it can achieve cell separation and concentration. At the same time, it also has a microscopic vibration amplitude, allowing cells to move in a very small range, achieving the separation of target cells and non-target substances, and can quickly and effectively separate bioactive substances with high throughput, especially separating some target cells from clinical or biological samples.

[0021] On the other hand, the present invention provides a method for separating target substances in biological fluid samples, which includes providing the aforementioned separation system, allowing the vibration power source to emit vibration waves of a certain frequency, so that the target substances are retained on the membrane and non-target substances pass through the membrane and flow out. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a three-dimensional schematic diagram (in an upright state) of a container provided with a membrane in a specific embodiment of the present invention.

[0023] Figure 2 It is a three-dimensional schematic diagram of a container provided with a membrane in a specific embodiment of the present invention (inverted state).

[0024] Figure 3 is a cross-sectional view of a container provided with a membrane in a specific embodiment of the present invention.

[0025] Figure 4 It is a three-dimensional schematic diagram of a separation system in a specific embodiment of the present invention (front view in assembled state).

[0026] Figure 5 This is a three-dimensional schematic diagram of a separation system in a specific embodiment of the present invention (back side in assembled state)

[0027] Figure 6 It is a cross-sectional schematic diagram of a separation system in a specific embodiment of the present invention.

[0028] Figure 7 It is a schematic diagram of the three-dimensional decomposition structure of a separation system in a specific embodiment of the present invention.

[0029] Figure 8 It is a comparison of the final cell residue on the membrane after separation and recovery of cells at a frequency of 50HZ with the final cell residue on the membrane without vibration filtration (Filter-4 means the micropore size is 4 microns, followed by 6 microns, 8 microns, 10 microns, and 12 microns).

[0030] Fig. 9 It is a schematic diagram of the principle structure of different separation methods. DETAILED DESCRIPTION

[0031] How the present invention is implemented is now described by way of examples. These are merely limited examples and do not violate the spirit of the principles of the present invention. These descriptions do not constitute any limitation to the present invention.

[0032] Vibration separation system

[0033] The separation system of the present invention, such as Figure 4-7As shown, it includes a support plate 60, on which there is a structure for fixing a membrane carrier container (generally metal or non-metal), and the membrane is a microporous array membrane. In some embodiments, one or more elastic elements are arranged below the support plate, and preferably there are four springs 32, 33, 34, 35, which are used to make the support plate stand up and have a relatively suspended position. One end of the spring can be connected to the support plate, and the other end is used to contact the operating table 40. In some embodiments, a vibration power source 20 is arranged on the support plate, and the vibration power source can cause vibration when powered on. The power source can be fixedly arranged on the support plate; for example Figure 4 As shown, the power source is suspended on the support plate through a suspension structure 201. Of course, one power source or multiple power sources are also possible, and 606 in the figure provides fixation for an additional power source (a small vibration source). When there is only one vibration motor, in order to maintain the stability of the support plate, some counterweights 605 can be added to allow the support plate to be in a relatively stable suspended state. Of course, other methods can be used to allow the support plate to be in a relatively stable, suspended state. In this way, when the power source vibrates, the energy can be stably transferred to the membrane through the support plate and springs, and the frequency of the vibration can be controlled more flexibly and precisely. On the contrary, if the support plate contacts many external objects, the vibration wave will be weakened or lost during transmission, and finally the vibration wave actually received on the membrane will be very weak and cannot have the effect of depolarizing the membrane surface. In some embodiments, the support plate includes a fixed film carrier container which may have a hole 603, so that the film carrier container is located in the hole. At the same time, a clamp 30 is used to fix the container so that it is fixed on the support plate. First, it can stably receive vibration waves from the support plate. In addition, after the liquid in the container passes through the membrane, it can flow under the support plate, and the outflowing liquid can be released into the waste liquid collection container. The setting of the clamp 30 can be in a variety of ways. For example, two limit blocks 301 and 302 matching the container are located on both sides of the container to achieve the effect of limiting and fixing. When it is necessary to unload the container, the clamp can be flexibly operated to facilitate the installation and unloading of the container.

[0034] In some embodiments, the film-carrying container 10 generally includes a cavity 103, and the membrane 105 is disposed at the bottom of the cavity. The liquid sample is collected in the cavity 101 and located above the membrane 105. The liquid can pass through the membrane and flow into the other side of the membrane (such as Figure 1-3As shown). In some embodiments, the container includes a base or a connecting seat 102, on which a snap structure 109, 110 is arranged, and the snap structure is connected to another container below. The container can be called a negative pressure container 11, and the negative pressure container includes an opening, and a snap structure 112, 111 is arranged around the opening. The snap structure cooperates with the snap structure of the base, so that the negative pressure container 11 is connected to the base 107. The base 102 can be an annular structure 107 surrounding a plane 106, and the opening and the peripheral structure of the negative pressure container are equivalent to the plane of the base, so that when the negative pressure container is connected to the base, the opening and the peripheral structure of the negative pressure container can be just snapped on the plane, and the opening of the negative pressure container contacts the bottom of the membrane, thereby forming an airtight sealing structure. In this way, when the negative pressure container has liquid, for example, the lower surface of the membrane has air or liquid, when the liquid drops with gravity, the air pressure on the lower surface of the membrane and the upper surface of the descending liquid drops, forming a negative pressure, thereby driving the liquid on the membrane to flow downward through the membrane. Of course, a vacuum pump may be connected to the lower opening of the negative pressure container, thereby forming negative pressure to drive the liquid on the membrane to flow to the bottom of the membrane through the membrane pores.

[0035] like Figure 4-5 is a schematic diagram of the structure in which the film-carrying container 10 is fixedly arranged on the support plate, and Figure 7 This is a three-dimensional exploded view of the structure. Such a structure can separate special substances in the sample on the membrane, such as tumor cells, etc.

[0036] Example 1: Effects of different frequencies on cell separation flux and damage .

[0037] 3 million human lymphoblastoid cells (T2) in 5 mL PBS buffer. The filter cup in the device is equipped with a Parylene filter membrane (not easy to deform) with a hexagonal micropore array of 1 million inscribed circles with a diameter of 6.9 μm and a membrane thickness of 10 μm. The estimated number of cells intercepted on the membrane is 3 times the number of micropores. Figure 4-6 After the vibration of the system (in the absence of the negative pressure container 11), the 5 mL T2 cell suspension is added to the filter cup for separation, and the total filtration time is measured to obtain the separation flux. Under the same experimental conditions, 5 mL PBS suspension containing 3 million human chronic myeloid leukemia cells (K562) and human acute T lymphocytes (Jurket) are used as samples to be separated, and the statistical separation flux is shown in the following table.

[0038]

[0039] Note: Statistics of cell damage were performed by DAPI fluorescent staining of cells on the membrane after separation, and it was found that there was fragmentation and some nuclear fragmentation.

[0040] The conventional vortex (vortex instrument) method is to fix the film-carrying container of the present invention on the vortex instrument, and the results are as follows:

[0041]

[0042] Through our experiments, under the same 50HZ vibration frequency, for the separation of the same sample, the survival rate of the target cells was only less than 50% when the vortex method was used for membrane separation. Although most of the cells were separated, they were damaged to varying degrees.

[0043] Compared with the first example, the separation effect with only the support plate (the support plate is placed directly on the operating table) and no spring is as follows:

[0044]

[0045] As can be seen from the above experimental data, as the frequency of vibration increases, the separation flux increases, and the efficiency increases, but as the frequency increases, different cells have the ability to withstand different damage. For T2 samples and K562 samples, when the frequency is 333 HZ, the cells remain intact and rarely damaged. However, for human acute T lymphocytes, the target cells separated under high-frequency vibration are seriously damaged. This shows that the system of the present invention, when confirming the analysis of cells, needs to clarify what type of target cells are, and then select the appropriate frequency to vibrate according to the target cells. If the spring of the present invention is not selected, and only the support plate is used, when the vibration frequency is increased, the separation flux is reduced, and at the same time, damage to cells to varying degrees is caused. It can be seen that the support plate has an important role with a spring, especially at high frequencies, for the damage of cells to play a particularly important purpose.

[0046] As mentioned above, in each micropore, due to excessive cells blocking the micropores, under the action of capillary action and the pressure difference across the membrane (simply relying on the pressure difference or gravity), although a small amount of liquid will still flow to the bottom of the membrane, the precise separation function of the membrane cannot be implemented and the concentration efficiency will be very low.

[0047] When the vibration power is used, the support plate is affected by the uniform surface vibration wave from the spring system, and the vibration is transmitted to the membrane, causing the target cells (tumor, T cells, human lymphoblastoid cells (T2)) and other components on the membrane to be affected by the vibration wave, leaving the position on the membrane surface, randomly rearranged with the components above and around, and re-screened by the micropores with other non-target components, so that the non-target components flow to the other side of the membrane through the micropores, while the target cells remain on the membrane. The present invention has a spring, and the entire vibration platform is placed on the spring assembly. The spring assembly has two functions, one is to isolate the vibration system from the external mechanical connection to avoid energy loss; the other more critical function is to convert the vibration generated by the point-like vibration source into a planar vibration source parallel to the micropore array membrane as much as possible through the spring system, so that the vibration can be more efficiently and uniformly transmitted to the separation system and the membrane surface. Therefore, the entire spring system plays the role of vibration isolation and vibration converter, which not only reduces unnecessary vibration energy loss, but also can appropriately suppress the macroscopic displacement in the horizontal direction, and better apply the energy evenly to the membrane plane, thereby overcoming the problem that in the case of high frequency, if accompanied by high amplitude, the inner wall of the filter cavity may have too much impact on the liquid, and even cause macroscopic vortexes, thereby causing damage to the local target cells. Generally, it is hoped that a relatively low amplitude can be generated in the case of high frequency to achieve separation while maximizing the activity of cells. Therefore, the designed spring system can improve the efficiency of the vibration filtration system while avoiding the generation of large macroscopically uneven local high shear force flow fields, ensuring the efficiency of the cell separation system while avoiding damage to the cells by local high shear forces.

[0048] Example 2: Effect of vibration frequency on separation flux

[0049] 1 million T2 cells (total) in 5 mL PBS buffer, Figure 5-6 The device is equipped with a polycarbonate (PC) microporous array filter membrane with 400,000 8-micron diameter circular holes. The number of cells is 2-3 times the number of micropores (negative pressure is applied under the negative pressure container 11). The negative pressure is 10kPa, and the filter is vibrated while filtering. The total filtration time is measured to obtain the separation flux.

[0050]

[0051] Example 3: Effect of vibration frequency on cell retention rate

[0052] 3 million Chinese hamster ovary cells (CHO cells, cell diameter 14-20μm) (total) in 5mL PBS buffer, in the device shown above, respectively, equipped with a polyparaxylene (Parylene) microporous array filter with 1 million regular hexagonal holes with inscribed circle diameters of 6, 8.6 and 10.4μm, the number of cells is 3-4 times the number of micropores, and the number of cells before and after filtration is compared while vibrating. The effect of cell removal is very good at all frequencies, but this indicator is also related to the size of the micropores themselves. When the cells are close to the pore diameter, vibration may cause the cells that would otherwise be trapped on the membrane to deform more easily through the micropores. The CHO cell filtration with the regular hexagonal microporous array membrane with inscribed circle diameters of 8.6μm and 10.4μm under high-frequency vibration will have some small-sized and highly deformable cells passing through the filter membrane. The cell size in the filtrate was measured and it was found that the cell diameters were 14.7 ± 0.3μm and 15.4 ± 0.1 μm, respectively. If 10 micron non-deformable polystyrene microspheres (PS material) are used, and the same conditions are used for separation using a Parylene microporous array filter membrane with regular hexagonal holes of 8.6 μm inscribed circle diameter under 120HZ vibration conditions, 100% of the cells are intercepted. Therefore, under vibration conditions, cells larger than the micropore size pass through the pores mainly through their own deformation, rather than because the flexible microporous film deforms and distorts the aperture under vibration conditions. This proves that the vibration system guarantees the separation accuracy of the ultra-low pressure microporous array membrane.

[0053] Implementation Example 4: Effect of vibration frequency on cell recovery rate

[0054] 3 million K562 cells (total) in 5 mL PBS buffer, Figure 5-6 The device is equipped with a polyimide (PI) micropore array filter membrane with 1 million square holes with an inscribed circle diameter of 8.0 microns. The number of cells is 3-4 times the number of micropores. It is filtered while vibrating. After filtration, the membrane and the cells on the membrane are placed in the release liquid, and the ratio of recovered cells to original cells is calculated.

[0055] High-frequency vibration makes it difficult for cells to get stuck or adhere to the membrane. In some applications, it is necessary to resuspend and recover the cells trapped on the membrane. For example, for CHO cells, Figure 8 Middle C and Figure 8 Compared with Figure F, the micropores on the micropore array filter membrane are all 8μm regular hexagonal holes. Figure 8 As shown in the figure above, most of the cells on the membrane can be resuspended and recovered after membrane vibration-assisted separation, and only a very small number of cells stick to the membrane or get stuck in the mold holes; in the absence of vibration, such as Figure 8As shown in F, most of the cells adhere to the membrane or are stuck in the membrane pores and cannot be resuspended and recovered. Low recovery rate means that the cells are stuck in the pores and cannot be detached from the pores through vibration. At extremely high frequencies, there will be a decrease in recovery rate due to excessive shear force and excessive energy received per unit time, which may cause cell breakage.

[0056] Implementation Example 5: Effect of vibration on cell size screening using microporous array membranes with different pore sizes

[0057] 3 million CHO cells (total) in 5 mL PBS buffer. Figure 5-6 The device is equipped with a regular hexagonal Parylene microporous array filter membrane with 1 million pores of different diameters. The number of cells is 3-4 times the number of micropores. It vibrates at 70HZ while filtering, and the average size (diameter) of cells trapped on membranes of different micropore sizes is counted.

[0058]

[0059] Vibration will change the pore function of microporous membrane separation to a certain extent. When the cell diameter is close to (equal to or substantially equal to) the micropore diameter, the cell will be squeezed through the pore. Therefore, when selecting the pore size parameters under vibration conditions, it is necessary to select a membrane with a pore size smaller than that of the target cell. This may be for those microporous

[0060] The vibration for the application of separating cells for recovery is also related to the pore size of the microporous array membrane. At a vibration frequency of 50HZ, when 2 million Chinese hamster ovary cells (CHO cells, cell diameter is 14-20μm) are placed in a regular hexagonal microporous membrane with a micropore diagonal of 4, 6, and 8μm, very few cells are stuck in the microporous membrane, and a small number of cells adhere to the surface of the microporous membrane ( Figure 8 A, Figure 8 B), the diameter of the cells is larger than the pore size of the microporous membrane. However, on the 10 and 12 μm microporous membranes, there are cases where residual cells on the membrane surface are stuck in the micropores after vibration separation ( Figure 8 D, Figure 8 E), this is because vibration will change the pore size of the membrane itself, generally slightly larger, so the target cells are stuck in the pores. Being stuck in the pores is not convenient for separating the cells from the membrane, and it will also cause blockage of the membrane pores, making the cells stuck in the pores of the membrane and unable to achieve effective separation. The control group filtered CHO cells without vibration, and the cells remained on the membrane, making it difficult to release and recover them easily, and the separation effect was not achieved (experimental data omitted).

[0061] Finally, it should be noted that the above-described embodiments are only specific implementations of the present invention, which are used to illustrate the technical solutions of the present invention, rather than to limit them. The protection scope of the present invention is not limited thereto. Although the present invention is described in detail with reference to the above-described embodiments, ordinary technicians in the field should understand that any technician familiar with the technical field can still modify the technical solutions recorded in the above-described embodiments within the technical scope disclosed by the present invention, or can easily think of changes, or make equivalent replacements for some of the technical features therein; and these modifications, changes or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

[0062] The invention shown and described herein can be implemented in the absence of any element, limitation specifically disclosed herein. The terms and expressions used are used as illustrative terms rather than limiting, and it is not intended to exclude any equivalents of the features shown and described or parts thereof in the use of these terms and expressions, and it should be recognized that various modifications are feasible within the scope of the present invention. It should therefore be understood that, although the present invention is specifically disclosed through various embodiments and optional features, modifications and variations of the concepts described herein can be adopted by those of ordinary skill in the art, and it is believed that these modifications and variations fall within the scope of the present invention as defined in the appended claims.

[0063] The contents of the articles, patents, patent applications, and all other documents and electronically available information described or recorded herein are incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to incorporate into this application any and all materials and information from any such article, patent, patent application, or other document.

Claims

1. A system for separating a target substance in a biological fluid sample, the system comprising: A microporous array membrane is used to receive or contact a biological fluid sample; a vibration power source element capable of vibrating, and a transmission element that transmits the vibration emitted by the vibration power source element to the microporous array membrane, so that target substances in the biological fluid can be separated through the microporous array membrane; the transmission element includes a support plate for fixing the microporous array membrane, and a spring that allows the support plate to be in a suspended state and simultaneously converts and homogenizes the energy of the vibration source, the vibration power source is fixed on the support plate, and the microporous array membrane is arranged in a membrane carrier container.

2. The separation system according to claim 1, wherein: The microporous array membrane has 500-50,000,000 micropores / square millimeter per unit area, a micropore diameter of 0.1-50 microns, a membrane thickness of 1-100 microns, and a porosity of 5%-90%.

3. The separation system according to claim 1, wherein: The vibration power source element includes one or more of a coreless cup vibration motor and / or an eccentric wheel DC motor; an ultrasonic generator based on a transducer chip and / or a pneumatic vibration device.

4. The separation system according to claim 1, wherein: The spring is arranged under the support plate to support the support plate, so that the support plate is supported to stand by the spring.

5. The separation system according to claim 1, wherein: The membrane-carrying container includes the microporous array membrane and a cavity for receiving a biological liquid sample, so that the liquid sample in the cavity contacts the microporous array membrane.

6. The separation system according to claim 5, wherein: The support plate includes a structure for mounting the microporous array membrane container, through which the membrane container is detachably fixed on the support plate.

7. The separation system according to claim 5, wherein: The container comprises an ultra-low negative pressure element below, and the element is located below the microporous array membrane and is used to apply a certain negative pressure to the microporous array membrane.

8. The separation system according to claim 7, wherein the negative pressure element comprises a lower cavity, one end opening of which is snap-fitted to the base of the microporous array membrane carrier container, so that the microporous array membrane and the opening form an airtight seal, thereby forming a negative pressure between the lower cavity and the microporous array membrane.

9. The separation system according to claim 1, wherein: The target substance in the biological fluid sample is one or more of tumor cells, abnormal circulating cells, cell lines producing antibodies or antigens, fat cells, macrophages, fungal spores, bacteria, viruses, and extracellular vesicles.

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