An apparatus for progressively accelerating the centrifugal separation of fine particulate matter with an approximate sedimentation velocity

Abstract

The present invention provides a device for progressively accelerating centrifugal separation of fine particles with an approximate sedimentation velocity, which comprises a centrifuge bottle, a separation chamber, and a collection chamber sequentially installed. A slender tube is provided at the bottom of the centrifuge bottle, and a liquid flow channel is arranged between the slender tube and the bottom of the separation chamber. An overflow port communicating with the collection chamber is provided at the top of the separation chamber. The device of the present invention is installed on a centrifuge for centrifugal separation. Under the action of centrifugal force, the specimen liquid flows out of the centrifuge bottle controllably, is injected into the separation chamber from the bottom of the separation chamber, gradually pushes up the liquid level of the separation chamber in batches, and continuously pushes the particles with a slightly smaller sedimentation velocity to overflow into the collection chamber, realizing the separation of particles with an approximate sedimentation velocity. The structure of the present invention is simple, the cost is low, no density liquid is required compared with the gradient density method, the biological activity is well maintained, no ultra-long travel is required compared with ordinary centrifugal devices, the requirement for the centrifuge is low, the separation effect is good, and it is suitable for large-scale popularization and use.

Description

Technical Field

[0001] The present invention belongs to the technical field of fine particulate matter separation and purification, and specifically relates to a device for separating fine particulate matter with an approximate sedimentation velocity by progressive acceleration centrifugation. Background Art

[0002] The sedimentation velocity U of particulate matter suspended in a liquid in a centrifugal force field is U = Jr 2 (ρ 2 - ρ 1 )α, where U represents the sedimentation velocity of the particulate matter, J represents the sedimentation coefficient, and its specific value is related to the viscosity of the liquid, the concentration of the particulate matter, etc., r represents the radius of the particulate matter, ρ 2 represents the density of the particulate matter, ρ 1Indicates the density of the specimen liquid, and α indicates the centrifugal acceleration. A variety of particulate matters with different volumes and densities have different sedimentation velocities in the same liquid. When the sedimentation velocity ratio of two particulate matters is large, the two particulate matters can be separated by differential centrifugation. When the sedimentation velocity ratio of the two particulate matters is small, a larger sedimentation stroke is required for separation, then the size of the centrifuge is very large, and it becomes very difficult to maintain the balance of the centrifuge. Currently, ordinary centrifugation technology cannot meet this requirement. To solve this problem, technicians increase the liquid density to expand the sedimentation velocity ratio of the two particulate matters and thus separate them, that is, density gradient centrifugation technology. However, it has a poor separation effect on particles with similar densities, mainly due to volume differences resulting in certain differences in sedimentation velocity. And because the density liquid is expensive and the operation process is also relatively complex, the density gradient centrifugation method is mainly used for the separation of a small number of samples in the scientific research field. When extracting rectal epithelial cells from stool specimens, although impurities with a diameter of more than 50 μm can be removed through multi-stage filtration technology, there are still many impurities in the filtrate, including a large amount of food residues, Escherichia coli, and various crystals. The impurities account for more than 90% of the mass ratio of the formed components. These impurities have different densities (0.9 g / cm3 - 2.4 g / cm3) and large diameter differences (1 μm - 50 μm), covering the density range (1.05 g / cm3 - 1.1 g / cm3) and diameter range (10 μm - 20 μm) of rectal epithelial cells. Using the density method for separation takes a long time, is complex in operation, has a high economic cost, and the effect is not good. The elutriation rotor centrifuge uses the principle that the direction of centrifugal force is opposite to the direction of the liquid flow and can separate two particulate matters with relatively similar sedimentation velocities. Whether this sedimentation velocity difference is caused by density differences or volume differences, theoretically, rectal epithelial cells can also be separated from stool. However, the elutriation rotor centrifuge has a complex structure, a cumbersome operation, requires a special person to operate, and is expensive. Currently, it is rarely used and difficult to be put into clinical laboratory use. CN114574323A discloses a cell separation device that controls the clamp, delivery pipe, and drain pipe to move between the solution rack, magnetic rack, mixing mechanism, tube position, and centrifuge through a robotic arm, thereby realizing the automation of cell separation experiments, avoiding mistakes or errors caused by manual operation, improving the accuracy of separation experiments and the efficiency of cell separation, and ensuring the accuracy of test results. CN218089499U discloses a mesenchymal stem cell separation and extraction device. By setting a centrifugation mechanism, test tubes of different specifications can be fixed, and there is no need to replace the test tube rack. The operation is very simple and convenient, and it saves time relatively. By setting a cooling mechanism, the activity of mesenchymal stem cells is prevented from decreasing. However, neither of them can solve the problem of cell sedimentation velocity. Currently, there is no other device that can well complete the separation of fine particulate matters with certain differences but not very large differences. Therefore, it is a difficult problem in clinical laboratories to separate fine particulate matters with certain differences but not very large differences in a relatively economical, convenient, and large-scale manner. Summary of the Invention

[0003] The present invention aims to solve at least one of the technical problems existing in the prior art. For this purpose, the present invention provides a device for progressively accelerating centrifugal separation of fine particles with an approximate sedimentation velocity, which includes a centrifuge bottle 2, a separation chamber 3, and a collection chamber 4. The centrifuge bottle 2 is installed on the separation chamber 3; the separation chamber is installed on the collection chamber 4. A sealing cover 21 is provided at the upper part of the centrifuge bottle 2, and a slender tube 22 is provided at the bottom 23 of the centrifuge bottle. The slender tube 22 faces the separation chamber 3, and a liquid flow channel 38 is provided between the slender tube 22 and the bottom 35 of the separation chamber 3. An overflow port 33 communicating with the collection chamber is provided at the top of the separation chamber. The device for progressively accelerating centrifugal separation of fine particles with an approximate sedimentation velocity of the present invention is installed on a centrifuge for centrifugal separation. Under the action of centrifugal force, the specimen liquid flows out of the centrifuge bottle controllably and is injected into the separation chamber from the bottom of the separation chamber. A negative pressure is formed in the centrifuge bottle. By controlling the speed of the centrifuge in an acceleration-constant speed-acceleration-constant speed cycle, the centrifugal force is controlled, and the specimen liquid is thrown out into the separation chamber in batches and the liquid level of the separation chamber is gradually pushed up in batches. The particles with a slightly smaller sedimentation velocity are continuously pushed to the overflow port to overflow into the collection chamber, thereby realizing the separation of particles with an approximate sedimentation velocity.

[0004] Further, an inner bottle 24 is provided in the centrifuge bottle, and the inner cavity of the inner bottle is connected to the slender tube 22 through a bottle inner tube 29.

[0005] Further, an air chamber 28 communicating with the upper space of the centrifuge bottle is provided at the bottom of the inner cavity of the centrifuge bottle, and the air chamber is connected to the upper space of the centrifuge bottle through an air duct 5. By providing an air chamber at the bottom of the centrifuge bottle, the pressure change rate caused by the continuous ejection of the specimen liquid during the acceleration of the centrifuge can be appropriately reduced, so that the centrifuge can eject the specimen liquid without a too high rotational speed. At the same time, the length of the slender tube can be increased, which can better control the outflow of the specimen liquid, and also reduces the centrifugal radius and the load of the centrifuge.

[0006] Further, an inner tube 26 is provided in the inner bottle 24, and a perforation 27 is provided at the upper part of the inner tube.

[0007] Further, the end of the slender tube is a low-strength closed end, and a spike portion 36 is provided on the bottom 35 of the separation chamber corresponding to the end of the slender tube.

[0008] Further, a partition ring 37 is provided in the separation chamber. The partition ring is a channel structure with outwardly extending upper parts and two open ends. Its upper end face is higher than the overflow port, and its lower part is a central liquid channel 310. A liquid flow channel 38 is provided below the lower end face of the partition ring.

[0009] Furthermore, an inner wall of the separation chamber and an outer wall of the partition ring 37 define a separation cavity 311, and a middle part of the separation cavity is larger than its upper and lower parts.

[0010] Furthermore, an inner tube 39 is disposed in the central liquid passage 310.

[0011] Furthermore, a bottom of the inner tube is a blind end and is mounted above a bottom of the separation chamber, and an upper end is an open end. An upper end surface 312 of the inner tube is higher than a maximum cross-section of the separation cavity 311.

[0012] The device for progressively accelerating centrifugal separation of fine particles with an approximate sedimentation velocity of the present invention controls the centrifugal force by controlling its rotation speed, so that the specimen liquid in the centrifuge bottle enters the separation chamber orderly, and gradually pushes various particles in the separation chamber to move towards the outlet direction. By using the centrifugal force acting on the particles in the liquid pointing to the bottom of the separation chamber during uniform rotation, the particles settle towards the bottom of the separation chamber. The particles with a large sedimentation velocity move a large distance back in the same time, and the particles with a small sedimentation velocity move a small distance back in the same time. Therefore, after multiple pushes, the aggregation ranges of various particles with different sedimentation velocities are separated, and finally, they overflow the separation chamber along with the rising liquid level and reach the collection chamber. The device for progressively accelerating centrifugal separation of fine particles with an approximate sedimentation velocity of the present invention is simple. Compared with the elutriation rotor centrifugal device used in the elutriation rotor centrifugation method, the structure of the present invention is extremely simple, greatly saving the equipment procurement cost, and also saving a large amount of usage cost. Compared with the gradient density method, it does not require a density liquid as the cell suspension medium, and the biological activity is well maintained, saving a large amount of usage cost. Compared with the ordinary centrifugal device, it does not require an extremely long stroke, has low requirements for the centrifuge, and has a particularly good separation effect, being suitable for large-scale popularization and use. Description of the Drawings

[0013] Figure 1 is a schematic structural diagram of a device for progressively accelerating centrifugal separation of fine particles with an approximate sedimentation velocity of the present invention;

[0014] Figure 2 is a schematic diagram of the sealed volume V of the specimen liquid f diagram;

[0015] Figure 3 is a schematic diagram of the effective volume V of the separation chamber c diagram;

[0016] Figure 4 is V 1 indicating the volume of the specimen liquid thrown out;

[0017] Figure 5 is a schematic diagram of the volume V of the pushing unit 3 diagram;

[0018] Figure 6It is a schematic diagram of the distance from the maximum cross-section of the conical part of the separation chamber to the overflow port;

[0019] Figure 7 It is a schematic diagram of the state after adding the specimen liquid to the centrifuge bottle;

[0020] Figure 8 It is a schematic diagram of the structure of another device for progressive acceleration centrifugal separation of fine particles with an approximate sedimentation velocity;

[0021] Figure 9 It is a schematic diagram of the structure of yet another device for progressive acceleration centrifugal separation of fine particles with an approximate sedimentation velocity;

[0022] Among them, 1. Centrifuge rotating shaft; 2. Centrifuge bottle; 21. Sealing cover; 22. Elongated tube; 23. Bottom of the centrifuge bottle; 24. Inner bottle; 29. Tube inside the bottle; 25. Overflow trough; 26. Inner tube; 27. Perforation; 28. Air cavity; 3. Separation chamber; 31. Conical section; 32. Cylindrical section; 33. Overflow port; 34. Outer edge platform; 35. Bottom of the separation chamber; 36. Spiked part; 37. Partition ring; 38. Liquid flow channel; 39. Tube inside the chamber; 310. Central liquid channel; 311. Separation cavity; 312. Upper end face of the tube inside the chamber; 4. Collection chamber; 5. Air channel. Detailed implementation manners

[0023] The embodiments of the present invention will be described in detail below. The examples of the embodiments are shown in the drawings, where the same or similar reference numerals denote the same or similar elements or elements with the same or similar functions from beginning to end. The embodiments described below by referring to the drawings are exemplary and are only used to explain the present invention and should not be construed as a limitation to the present invention.

[0024] In the description of the present invention, it should be understood that the orientation or positional relationship indicated by the terms "center", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to the present invention.

[0025] In the description of the present invention, it should be noted that unless otherwise clearly specified and limited, the terms "installed", "connected", "connected" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be directly connected, or indirectly connected through an intermediate medium, and can be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0026] This embodiment is an invention directed to fine particulate matter, especially fine particulate matter at the cellular level. Terms such as "a certain difference", "larger", and "smaller" in this article also refer to fine particles such as those in cytology, rather than the vocabulary terms described in machinery or daily life.

[0027] As Figure 1 Shown is a device for progressively accelerating centrifugal separation of fine particulate matter with an approximate sedimentation velocity according to the present invention, including a centrifuge, a centrifuge bottle 2, a separation chamber 3, and a collection chamber 4. The centrifuge is provided with a centrifuge rotating shaft 1; one end of the centrifuge bottle 2 is provided with a sealing cap 21, and the other end is the bottom 23 of the centrifuge bottle. A slender tube 22 communicating with the inner cavity of the centrifuge bottle is provided on the bottom 23 of the centrifuge bottle; the separation chamber 3 includes an upper part and a lower part that are connected. The upper part is a cylindrical section 32, and the lower part is a conical section 31. The cylindrical section 32 is connected to the large end of the conical section 31. The other end of the cylindrical section 32 is an open end. The small end of the conical section 31 is a closed bottom 35 of the separation chamber. The bottom 35 of the separation chamber can be a pointed bottom or a flat bottom. An outer edge platform 34 is provided at the opening of the cylindrical section. A number of overflow ports 33 communicating the inside and outside of the separation chamber 3 are provided near the open end of the cylindrical section 32. The overflow ports 33 can be through holes or pipes opened on the steps provided between the outer edge platform and the cylindrical section, or openings or pipes opened on the tube wall near the opening of the cylindrical section; the collection chamber 4 is an open container. The outer edge platform 34 of the separation chamber 3 is installed at the open end of the collection chamber 4, and the bottom 35 of the separation chamber faces the collection chamber 4; the outer edge platform 34 of the separation chamber is connected to the bottom 23 of the centrifuge bottle. The slender tube 22 of the centrifuge bottle is inserted into the separation chamber and points to the bottom 35 of the separation chamber, but the end of the slender tube 22 of the centrifuge bottle does not touch the bottom 35 of the separation chamber. The centrifuge bottle 2, the separation chamber 3, and the collection chamber 4 are combined into a centrifugal kit and fixed in a hanging basket on the centrifuge rotating shaft 1 and rotate with the centrifuge rotating shaft 1.

[0028] After the centrifuge bottle 2, the separation chamber 3, and the collection chamber 4 are combined into a centrifugal kit, the interiors of the separation chamber 3 and the collection chamber 4 are both in communication with the atmosphere.

[0029] In another embodiment, as Figure 8As shown, an air chamber 28 communicating with the upper space of the centrifuge bottle is provided at the inner bottom of the centrifuge bottle 2. The air chamber 28 communicates with the upper space of the centrifuge bottle through an air passage 5. In this embodiment, an inner bottle 24 is arranged inside the centrifuge bottle 2. There is a gas passage 5 between the outer wall of the inner bottle 24 and the inner wall of the centrifuge bottle 2. A bottle inner tube 29 is provided at the bottom of the inner bottle 24. The inner cavity of the inner bottle 24 is communicated with the slender tube 22 of the centrifuge bottle 2 through the bottle inner tube 29. An air chamber 28 is formed between the outer wall of the bottle inner tube 29 and the inner wall of the centrifuge bottle 2. The air chamber 28 is kept sealed with the slender tube 22. The air chamber 28 communicates with the upper space of the centrifuge bottle through the air passage 5. By arranging an air chamber at the bottom of the centrifuge bottle, without increasing the overall height of the centrifuge bottle, the liquid level height inside the centrifuge bottle can be increased, and the hydraulic change rate caused by the continuous ejection of the specimen liquid can be appropriately reduced, so that the centrifuge can eject all the specimen liquid in the centrifuge bottle without a too high rotation speed.

[0030] An inner tube 26 is arranged inside the inner bottle 24 of the centrifuge bottle 2. The inner tube is a hollow tube with both ends open. A perforation 27 is provided at the upper part of the inner tube. There is a gap of about 1 mm between the lower end face and the inner wall of the bottom of the inner bottle 24. When the liquid level is lower than the perforation 27, the liquid outside the inner tube 26 will flow into the inner tube from the lower gap of the inner tube 26 when the centrifuge accelerates, thus forming an inward liquid flow from the outside to the inside at the bottom of the inner bottle 24 to prevent particulate matter from adhering to the bottom of the inner bottle. An annular overflow groove 25 is provided at the upper part of the inner tube 26. The inner ring of the overflow groove is integrally formed with the inner tube. The outer wall of the outer ring of the annular overflow groove 25 forms a seal with the inner wall of the inner bottle 24. There is a ventilation passage between the upper end of the inner tube 26 and the sealing cap 21, so that the inner cavity of the inner tube 26 is communicated with the air chamber 28. The opening of the inner tube 26 is small, and the annular groove is sealed with the inner cavity of the inner bottle 24. During use, the specimen liquid is added until liquid overflows from the upper mouth of the inner tube, and then the residual liquid in the annular groove is sucked out, so as to ensure the accuracy of the added amount of the specimen liquid.

[0031] A partition ring 37 is provided in the separation chamber. The partition ring 37 is an annular ring with both ends open and the upper mouth flaring outwards. The upper mouth end face is higher than the overflow port of the separation chamber, the outer diameter of the upper mouth is smaller than the inner diameter of the cylindrical section of the separation chamber, and there is a liquid flow channel 38 below the lower mouth end face. The outer wall of the partition ring 37 and the inner wall of the separation chamber enclose a separation cavity 311. The middle cross-sectional area of the separation cavity 311 is the largest, and the cross-sectional area gradually decreases from the middle to both ends. The middle and lower part of the inner cavity of the partition ring is a central liquid channel 310. The opening of the slender tube 22 of the centrifuge bottle is located above the central liquid channel 310 and higher than the maximum cross-section of the separation cavity 311. When the rotational speed of the centrifuge decreases, the specimen liquid in the separation cavity 311 will flow back into the centrifuge bottle through the liquid flow channel 38 and the central liquid channel 310. When the liquid level in the separation cavity 311 is flush with the opening end face of the slender tube 22 of the centrifuge bottle, the liquid in the separation cavity 311 stops flowing. Since the cross-sectional area above the maximum cross-section of the separation cavity 311 gradually decreases, the amount of liquid flowing back from the separation cavity is very small, so that the existing orderly distribution state of the particulate matter in the separation cavity does not change significantly. Using this feature, the specimen liquid can be sucked back into the centrifuge bottle to perform a backwash on the centrifuge bottle, so that the cells attached to the bottom inner wall of the centrifuge bottle are detached and re-enter the specimen liquid, thereby increasing the cell separation amount of the specimen liquid. It can also allow air to be inhaled into the centrifuge bottle through this feature to reduce the rotational speed of the centrifuge.

[0032] In yet another embodiment, as Figure 9 shown, different from the previous embodiment, the taper of the conical section 31 of the separation chamber is reduced to obtain a better lifting effect, and the inner diameter of the central liquid channel 310 at the lower part of the partition ring 37 is increased. In order to reduce the residual liquid after separation, an inner tube 39 is provided in the central liquid channel 310. The outer diameter of the inner tube 39 is smaller than the inner diameter of the central liquid channel 310. The upper end of the inner tube 39 is open, and the lower end is a blind end and is arranged above the bottom of the separation chamber. The end of the slender tube 22 is a low-strength closed end. The slender tube extends into the inner cavity through the upper end opening of the inner tube 39, but the end face of the slender tube does not contact the bottom of the inner tube 39. A spike portion 36 is provided at the bottom of the inner tube 39 corresponding to the end of the slender tube, which is convenient for piercing the end closing portion of the slender tube when the centrifuge bottle is installed on the separation chamber and opening the channel from the centrifuge bottle to the separation chamber. The upper end face 312 of the inner tube is higher than the maximum cross-section of the separation cavity 311. When the rotational speed of the centrifuge decreases, the liquid level in the separation cavity only drops to be flush with the upper end face 312 of the inner tube, so that the existing orderly distribution state of the particulate matter in the separation cavity does not change significantly.

[0033] This device is mainly used for centrifugal separation and collection of biological cells, especially in commercial applications such as extracting rectal epithelial cells from feces, separating lymphocytes from blood, separating pleural and peritoneal effusion cells, primary cell separation, sperm selection, etc.

[0034] In addition, we also provide a method for progressive acceleration centrifugal separation of fine particles with approximate sedimentation velocities. For the separation of fine particles with approximate sedimentation velocities, although the sedimentation velocities are similar, they are only approximate after all and not equal. There are still differences in sedimentation velocities, but the differences are relatively small. General centrifugal devices and methods cannot centrifugally separate them. In this embodiment, taking the separation of rectal epithelial cells from feces as an example:

[0035] The steps of separating rectal epithelial cells from feces by using the device for progressive acceleration centrifugal separation of fine particles with approximate sedimentation velocities are as follows:

[0036] Mix 3 - 5 g of fecal specimens with 20 - 25 ml of preservation solution to obtain a stock solution. Filter the stock solution through oscillation to obtain a specimen solution. Seal the outlet of the slender tube 22 of the centrifuge bottle 2, inject the specimen solution into the centrifuge bottle 2, and seal the mouth of the centrifuge bottle with the sealing cap 21. The capacity of the centrifuge bottle used in this experiment is 42 ml, the length of the slender tube is 30 mm, the inner diameter of the open end is 2.2 mm, and the amount of the specimen solution added to the centrifuge bottle is 20 ml. Feces contain rectal epithelial cells and impurities. Relatively speaking, the sedimentation velocity of rectal epithelial cells is smaller, and the sedimentation velocity of impurities with larger volume and density is larger. Of course, there are also impurities with sedimentation velocities extremely similar to those of rectal epithelial cells and impurities with even smaller sedimentation velocities.

[0037] Install the separation chamber 3 on the collection chamber 4, remove the seal on the opening of the slender tube 22 of the centrifuge bottle, insert the slender tube 22 of the centrifuge bottle into the separation chamber 3, and the effective volume V of the separation chamber C is 2.6 ml (as Figure 3 shown). Place the bottom of the centrifuge bottle on the outer edge platform 34 of the separation chamber 3, and then combine the centrifuge bottle 2, the separation chamber 3, and the collection chamber 4 into a centrifugation kit and fix it on the centrifuge shaft 1.

[0038] Start the centrifuge, and for the first acceleration, accelerate the centrifuge to the speed N 1 , so that V 1 of the specimen solution in the centrifuge bottle is thrown out into the separation chamber (as Figure 4 shown). V 1 is slightly less than the effective volume V of the separation chamber C . In this experiment, V 1 = 2.4 ml, V c = 2.6 ml, and then run at a constant speed of N 1 for T 1 time.

[0039] The rotational speed N of the centrifuge 1 is determined according to the following formula:

[0040]

[0041] N represents the rotational speed of the centrifuge. Correspondingly, N 1 represents the rotational speed of the centrifuge after the first acceleration, N 2 represents the rotational speed of the centrifuge after the second acceleration, N n represents the rotational speed of the centrifuge after the nth acceleration;

[0042] V 0 represents the volume of air inside the centrifuge bottle after adding the specimen liquid and sealing it. As Figure 7 shown;

[0043] V represents the volume of the specimen liquid that has been thrown out. Correspondingly, V 1 represents the volume of the specimen liquid that has been thrown out during the first acceleration, V 2 represents the volume of the specimen liquid that has been thrown out during the second acceleration, V n represents the volume of the specimen liquid that has been thrown out during the nth acceleration;

[0044] V f represents the amount of specimen liquid flowing out of the centrifuge bottle before the outlet end of the slender tube is submerged by the specimen liquid, that is, the sealing volume (as Figure 2 shown). K represents the centrifugal coefficient, which is calculated according to the following formula:

[0045]

[0046] where R is the real-time centrifugal radius, H is the real-time liquid level height inside the centrifuge bottle, ρ 1 is the density of the specimen liquid, and P represents the atmospheric pressure.

[0047] T represents the time when the centrifuge runs at a constant speed. Correspondingly, T 1 represents the time when the centrifuge runs at a constant speed after the first acceleration, T 2 represents the time when the centrifuge runs at a constant speed after the second acceleration, T n represents the time when the centrifuge runs at a constant speed after the nth acceleration; When the centrifuge runs at a constant speed, the cells and impurities in the separation chamber will move back. The back-moving distance of the cells is b. The back-moving distance of the cells when the centrifuge runs at a constant speed for the first time is b 1 The back-moving distance of the cells when the centrifuge runs at a constant speed for the second time is b 2 The back-moving distance of the cells when the centrifuge runs at a constant speed for the nth time is b n The back-moving distance of the impurities with a larger sedimentation speed is c. The back-moving distance of the impurities when the centrifuge runs at a constant speed for the first time is c 1 The back-moving distance of the impurities when the centrifuge runs at a constant speed for the second time is c 2 The back-moving distance of the impurities when the centrifuge runs at a constant speed for the nth time is c n . The time T when running at a constant speed after the first acceleration 1It should be ensured that the particulate matters with larger volume and density can settle below the maximum cross-sectional area of the conical part of the separation chamber, so that when the specimen liquid enters the separation chamber during the second acceleration, no particulate matters with larger volume and density overflow from the separation chamber into the collection chamber with the liquid. T 1 The time can be set to the maximum value based on past experience or obtained through trial and error experiments. Run at a constant speed for time T 1 The time should ensure that the impurities with a larger sedimentation speed move back a distance greater than L from the bottom of the separation chamber. L represents the distance between the maximum cross-sectional area of the conical part of the separation chamber and the overflow outlet (as Figure 6 shown).

[0048] For the second acceleration, accelerate the centrifuge to speed N 2 and fling out the specimen liquid in the centrifuge bottle by volume V 2 and ensure that a small amount of the specimen liquid overflows from the separation chamber into the collection chamber, and then run at a constant speed for time T 2 ; T 2 The time should ensure that when the specimen liquid enters the separation chamber for the third time, no impurities with larger volume and density overflow from the separation chamber into the collection chamber with the liquid. T 2 The time can be set to the maximum value based on past experience or obtained through trial and error experiments; the centrifuge speed N 2 is determined according to the following formula

[0049]

[0050] For the third acceleration, accelerate the centrifuge to speed N 3 and fling out the specimen liquid in the centrifuge bottle by volume V 3 and ensure that an amount of liquid equal to V 3 overflows from the separation chamber into the collection chamber, and then run at a constant speed for time T 3 ; Run at a constant speed for time T 3 The time should ensure that the cells with a smaller sedimentation speed (rectal epithelial cells) move back a distance b 3 less than the liquid column lifting distance a above the maximum cross-sectional area of the conical part of the separation chamber 3 , and the impurities with a larger sedimentation speed move back a distance c 3 greater than or equal to the liquid column lifting distance a 3 ;

[0051] a is the lifting distance, which is the liquid column height corresponding to the volume of the specimen liquid flung out above the maximum cross-sectional area of the conical part of the separation chamber, as Figure 5 shown. Correspondingly, the lifting distance during the second acceleration of the centrifuge is a 2 , and the lifting distance during the nth acceleration of the centrifuge is a n; When the lifting distance is a millimeters, the liquid at the largest cross-section of the conical part of the separation chamber advances a millimeters in the direction of the outlet, and the cells and impurities in the specimen liquid at this position are also advanced a millimeters in the direction of the outlet. The rotational speed N of the centrifuge 3 is determined according to the following formula

[0052]

[0053] Then the centrifuge is accelerated - run at a constant speed - accelerated - run at a constant speed... until it reaches N n , and after a total of n times (in this embodiment, all n ≥ 4) of accelerated operation, the centrifuge operation is finally stopped. The constant-speed operation for T n time allows the cells with a smaller sedimentation speed (rectal epithelial cells) to move back a distance b n less than the lifting distance a of the specimen liquid column n , and the impurities with a larger sedimentation speed move back a distance c n greater than or equal to the lifting distance a of the specimen liquid column n , finally retaining the fecal impurities with a larger sedimentation speed in the separation chamber and pushing the rectal epithelial cells with a smaller sedimentation speed through the overflow port to be separated into the collection chamber. The rotational speed N of the centrifuge n is determined according to the following formula

[0054]

[0055] Although we know the law of the sedimentation speed of particulate matter, calculating the actual sedimentation speed of various particulate matters is very complicated. In the experiment, we often adopt the "trial and error method" to evaluate the sedimentation time required for particulate matter; after liquid overflows from the separation chamber, that is, after the third acceleration, regardless of whether the lifting distances in each stage are equal, as long as T n = a n N 3 2 T 3 / N n 2 a 3 , it can make the "lifting / moving back" rate of various particulate matters equal, that is to say, the separation effect achieved by each "acceleration - constant speed" is the same. In this way, only by observing the experimental results and continuously adjusting T 3 , and calculating T 3 through T n , a relatively ideal separation effect can be obtained.

[0056] In this embodiment, for the convenience of control, the volume of the specimen liquid thrown out after the third acceleration is equal to V 3 , the lifting distance for each acceleration is equal, a n = a 3 , where n ≥ 4, the rotational speed N of the centrifuge nDetermined by the following formula:

[0057]

[0058] Wherein, N n represents the rotational speed after the nth acceleration (where n ≥ 4), and V 3 represents the liquid volume required to generate the lifting distance a above the maximum cross-section (as Figure 5 shown), that is, the volume of the liquid column section with a length of a, and a can take any value within the range of 0.1 - L mm.

[0059] In order to make the return distance of specific particulate matter in the separation chamber equal during uniform operation at each stage, then T n = N 3 2 T 3 / N n 2 .

[0060] Its working principle is as follows: After the centrifuge starts running, the specimen liquid is quickly thrown into the separation chamber through the slender tube, causing the liquid level in the separation chamber to gradually rise and first submerge the port of the slender tube. The specimen liquid in the centrifuge bottle gradually decreases, and the air volume in the centrifuge bottle continuously increases and gradually forms a negative pressure. When the centrifuge gradually accelerates to N 1 , the specimen liquid flowing out of the centrifuge bottle reaches near the outlet of the separation chamber. Then, it runs uniformly at a rotational speed of N 1 for T 1 seconds. The centrifugal force no longer increases, and the force of the negative pressure acting on the specimen liquid in the centrifuge bottle balances the centrifugal force received by the specimen liquid. The specimen liquid in the centrifuge bottle no longer flows out. The cells suspended in the specimen liquid in the separation chamber move back to the bottom of the separation chamber under the action of the centrifugal force. T 1 should be appropriately increased relative to T 2 , T 3 to make all the impurities in the upper liquid of the separation chamber move back below the maximum cross-section of the conical part of the separation chamber.

[0061] When the centrifuge accelerates to N 2 , V 2 specimen liquid enters the separation chamber and causes a small amount of liquid to overflow from the separation chamber. Then, it runs uniformly at a rotational speed of N 2 for T 2 time; T 2 should be appropriately increased to make all the impurities in the upper liquid of the separation chamber move back below the maximum cross-section.

[0062] When the centrifuge accelerates to N 3 , V 3 specimen liquid enters the separation chamber and pushes up the liquid column at the maximum cross-section by a 3 . Then, it runs uniformly at a rotational speed of N 3 for T 3The return distance b of rectal epithelial cells with smaller time, density and volume 3 is less than a 3 , and the return distance c of impurities with larger density and volume 3 is greater than a 3 , the centrifuge continues to accelerate. Since the separation chamber is already completely filled with liquid, an equal amount of specimen liquid entering will cause an equal amount of liquid to be pushed out. During the subsequent n acceleration processes, the pushing distance is a n , the specimen liquid near the outlet of the separation chamber continuously flows into the collection chamber through the overflow port under the push of the specimen liquid being thrown out. The specimen liquid flowing into the collection chamber is the uppermost preservation liquid, cells, and impurities smaller in diameter than the cells. The return distance c of impurities with a larger sedimentation velocity is greater than the pushing distance a and is thus always restricted below the maximum cross-section of the conical part of the separation chamber and will never be pushed out of the separation chamber; the return distance of rectal epithelial cells with a smaller sedimentation velocity is less than the pushing distance. During each cycle of acceleration and uniform motion, the position of the rectal epithelial cells in the separation chamber rises by a certain distance and is finally pushed out of the separation chamber.

[0063] When the centrifuge runs at a uniform speed of N n rotations per minute, the return velocity U of the particulate matter in the centrifugal force field is proportional to the magnitude of the centrifugal force, and the return velocity U = Jr 2 (ρ 2 -ρ 1 ). The magnitude of the sedimentation movement coefficient J is related to the liquid viscosity, particulate matter density, etc. Since all particulate matters are in the same liquid system, for all particulate matters, the J value is equal. What determines their velocity difference is their own density and volume. Particulate matters with larger density and volume have a faster return velocity, while those with smaller density and volume have a slower return velocity. The return distance is equal to the product of the return velocity and the return time. Within the time T n , particulate matters with different sedimentation velocities have different return distances, thus separating particulate matters with different sedimentation velocities. In this way, most of the impurities can be separated to obtain a specimen liquid with fewer impurities.

[0064] In this embodiment, V 0 = 22 ml, V 1 = 2.4 ml, V f = 0.1 ml, R = 11.6 cm, H = 6.4 cm, ρ 1 = 1.015, N 1 = 355 revolutions per minute, T 1 = 200 seconds. When the rotational speed reaches N 1 , the specimen liquid thrown out of the centrifuge bottle just reaches near the lower part of the overflow port.

[0065] N 2 = 372 revolutions per minute, T 2 = 100 seconds. When the rotational speed reaches N2 A small amount of liquid flows out of the overflow port at this time.

[0066] N 3 = 388 revolutions per minute, T 3 = 52 seconds. When the rotational speed reaches N 3 the volume V of the lifting unit 3 = 0.25 ml, the lifting distance a = 1 mm, and the backward movement distance of the epithelial cells is less than 0.9 mm.

[0067] In this embodiment, for the convenience of control, since N 3 afterwards, the volume of the liquid thrown out each time is equal, and the distance a by which the particulate matter in the separation chamber is lifted each time the centrifuge accelerates is equal.

[0068] After 10 - 20 lifting and backward movement cycles, the rectal epithelial cells (with a diameter of 10 um - 20 um), Escherichia coli (with a diameter of 5 um - 8 um), and particulate matter with a relatively small diameter (<20 um) with a relatively small sedimentation velocity are gradually lifted to the outlet of the separation chamber and finally overflow into the collection chamber, while the particulate matter and various crystals with a larger diameter and density (density 1.5 g / cm 3 - 2.4 g / cm 3 ) are retained in the separation chamber.

[0069] After the above separation process is completed, the separated liquid in the collection chamber is subjected to a second separation experiment according to this method, so that the rectal epithelial cells are retained in the separation chamber, while the Escherichia coli and particulate matter with a diameter <10 um are separated into the collection chamber. Through two separations, we separate off most of the impurities, and only those impurities with a sedimentation velocity basically consistent with that of the rectal epithelial cells are mixed in the separated specimen, meeting the requirements of observation and diagnosis.

[0070] According to the same technical principle, we can also separate single cell components from blood, separate target cells from primary cell culture media, separate specific cells from pleural and peritoneal effusions, remove white blood cells and dead cells from semen, and separate other various particulate matters with certain differences in sedimentation velocity.

[0071] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and purposes of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A device for progressively accelerating centrifugal separation of fine particles with an approximate sedimentation velocity, characterized in that, it includes a centrifuge bottle (2), a separation chamber (3), and a collection chamber (4). The centrifuge bottle (2) is installed on the separation chamber (3); the separation chamber is installed on the collection chamber (4). A sealing cover (21) is provided at the upper part of the centrifuge bottle. A slender tube (22) is provided at the bottom (23) of the centrifuge bottle. The slender tube (22) faces the separation chamber (3). A liquid flow channel (38) is provided between the slender tube (22) and the bottom (35) of the separation chamber. An overflow port (33) communicating with the collection chamber is provided at the top of the separation chamber (3); a partition ring (37) is provided in the separation chamber. The partition ring has a structure with the upper part flaring outwards and both ends open. Its upper end face is higher than the overflow port. Its lower part is a central liquid channel (310). A liquid flow channel (38) is provided below the lower end face of the partition ring; a separation cavity (311) is formed between the inner wall of the separation chamber (3) and the outer wall of the partition ring (37). The middle part of the separation cavity is larger than its upper and lower parts; an inner bottle (24) is provided in the centrifuge bottle. The inner cavity of the inner bottle is connected to the slender tube (22) through a tube inside the bottle (29).

2. The device for progressively accelerating centrifugal separation of fine particles with an approximate sedimentation velocity according to claim 1, characterized in that, an air cavity (28) communicating with the upper space of the centrifuge bottle is provided at the bottom of the inner cavity of the centrifuge bottle. The air cavity is connected to the upper space of the centrifuge bottle through an air duct (5).

3. The device for progressively accelerating centrifugal separation of fine particles with an approximate sedimentation velocity according to claim 1, characterized in that, an inner tube (26) is provided in the inner bottle (24). A perforation (27) is provided at the upper part of the inner tube.

4. The device for progressively accelerating centrifugal separation of fine particles with an approximate sedimentation velocity according to claim 1, characterized in that, the end of the slender tube is a low-strength closed end. A spike part (36) is provided on the bottom (35) of the separation chamber corresponding to the end of the slender tube.

5. The device for progressively accelerating centrifugal separation of fine particles with an approximate sedimentation velocity according to claim 1, characterized in that, a tube inside the chamber (39) is provided in the central liquid channel (310).

6. The device for progressively accelerating centrifugal separation of fine particles with an approximate sedimentation velocity according to claim 5, characterized in that, the bottom of the tube inside the chamber is a blind end and is erected above the bottom of the separation chamber. The upper end is an open end. The upper end face (312) of the tube inside the chamber is higher than the maximum cross-section of the separation cavity (311).

Images