A butterfly microfluidic chip

Abstract

The present invention relates to the technical field of medical devices, and specifically discloses a butterfly microfluidic chip, which includes a chip body. A rotating shaft clamping position is provided at the center of the circle of the chip body. A first sample processing area, a second sample processing area, a sorted sample area, and a final sample distribution area are respectively arranged on the chip body. Among them, the first sample processing area is connected to the second sample processing area through a pipeline, the second sample processing area is connected to the sorted sample area through a pipeline, the sorted sample area is connected to the final sample distribution area through a pipeline, the final sample distribution area is semi-circular and arranged around the rotating shaft clamping position, and the distance between the second sample processing area and the rotating shaft clamping position is greater than the distance between the first sample processing area and the rotating shaft clamping position. The present invention eliminates the cumbersome steps of manually extracting PBMCs, reduces the operation difficulty, and at the same time ensures the purity of PBMCs, avoids the loss of target cells, and can improve the extraction efficiency of PBMCs.

Description

Technical Field

[0001] The present invention relates to the technical field of medical devices, and particularly to a butterfly microfluidic chip. Background Art

[0002] The main cell types of peripheral blood mononuclear cells (PBMCs) in peripheral blood are cells with a single nucleus in the blood, mainly including lymphocytes (T / B), monocytes, phagocytes, dendritic cells and a small number of other cell types. Since the number of red blood cells and platelets in whole blood is huge, the presence of these cells will have a great impact on antibody labeling and flow analysis, and it is very likely to obtain incorrect results. This leads to the fact that when directly using whole blood to detect lymphocytes or monocytes clinically, the experimental results obtained are generally not recognized. And the various subsets of lymphocytes maintain a certain proportion and quantity in the normal body. Clinically, it is found that when this proportion is disordered, the immune function in the body may be disordered, which will lead to the occurrence of diseases. By immunotyping (qualitative) and quantitative analysis of lymphocytes in the blood, the immune function of the collective can be evaluated more accurately, which is of great significance for the clinical diagnosis of immunodeficiency diseases, autoimmune diseases and guiding immunotherapy. Therefore, in the laboratory, PBMCs are commonly extracted from whole blood to simulate the in vitro blood immune environment for experiments, so it is necessary to extract the mononuclear cells for detection.

[0003] Peripheral blood mononuclear cells include lymphocytes and monocytes, etc., and their volume, morphology and density are different from other cells. The density of red blood cells and granulocytes in the blood is relatively large, about 1.090 g / ml, but the density of lymphocytes and monocytes is 1.075 - 1.090 g / ml, and that of platelets is 1.030 - 1.035 g / ml. After appropriate layering treatment, with the action of centrifugation and separation liquid, cells with a relatively large density such as red blood cells and granulocytes will settle at the bottom. At this time, white blood cells are still in the upper layer of the separation liquid and in the separation liquid layer (a small number), and substances with the lowest density such as platelets are in the uppermost layer. Finally, the PBMC layer is aspirated with a pipette to obtain the PBMC layer mixed solution for laboratory use.

[0004] Currently, for the extraction of PBMCs, after density gradient centrifugation, it is necessary to observe the position of the PBMC layer by the naked eye and manually aspirate the PBMCs. The purity of PBMCs is low. This operation requires a high level of training and needs to practice this operation repeatedly, which is not conducive to the popularization of this method; moreover, the extracted PBMCs have complex components, and for downstream analysis, operations such as washing are also required to remove the separation liquid or other unnecessary media, increasing the probability of target cell loss. Summary of the Invention

[0005] The technical problem to be solved by the present invention is: how to avoid the defects of low purity of target cells PBMC extracted manually and complex operation.

[0006] To solve the above technical problem, the present invention provides a butterfly microfluidic chip, including a chip body. A shaft clamping position is provided at the center of the circle of the chip body. A first sample processing area, a second sample processing area, a sorted sample area, and a sample final distribution area are respectively arranged on the chip body. Among them, the first sample processing area is connected to the second sample processing area through a pipeline, the second sample processing area is connected to the sorted sample area through a pipeline, the sorted sample area is connected to the sample final distribution area through a pipeline, the sample final distribution area is semi-circular and arranged around the shaft clamping position, and the distance between the second sample processing area and the shaft clamping position is greater than the distance between the first sample processing area and the shaft clamping position.

[0007] In some embodiments, the first sample processing area includes:

[0008] A sample mixing and temporary storage cavity. A sampling hole is provided at one end of the sample mixing and temporary storage cavity close to the center of the circle of the chip body. A blocking area is provided at the other end of the sample mixing and temporary storage cavity away from the center of the circle of the chip body. The blocking area and the sampling hole are diagonally arranged on the sample mixing and temporary storage cavity; and

[0009] A first ball. The first ball is arranged inside the sample mixing and temporary storage cavity and always moves along the inner wall of the sample mixing and temporary storage cavity at the end away from the center of the circle of the chip body.

[0010] In some embodiments, a first exhaust hole is further provided on the sample mixing and temporary storage cavity. The first exhaust hole and the blocking area are on the same side, and the distance between the first exhaust hole and the center of the circle of the chip body is less than the distance between the blocking area and the center of the circle of the chip body.

[0011] In some embodiments, the diameter of the first ball is less than one-third of the depth of the sample mixing and temporary storage cavity.

[0012] In some embodiments, the blocking area includes:

[0013] A convex structure. The convex structure is arranged on the inner wall of the sample mixing and temporary storage cavity away from the center of the circle of the chip body;

[0014] A first shallow platform structure. The first shallow platform structure is arranged inside the sample mixing and temporary storage cavity and on one side of the convex structure;

[0015] A wax valve structure. The wax valve structure is arranged outside the sample mixing and temporary storage cavity and corresponds to the first shallow platform structure;

[0016] A deep platform structure, which is arranged on the side of the wax valve structure away from the first shallow platform structure; and

[0017] A second shallow platform structure, which is arranged corresponding to the deep platform structure, and the second shallow platform structure is provided with a first thin tube for guiding the sample mixing medium to the second sample processing area.

[0018] In some embodiments, the depth of the first shallow platform structure is less than the depth of the sample mixing and temporary storage cavity, and the depth of the first shallow platform structure is greater than the height of the convex structure. The distance between the bottom surface of the convex structure and the bottom surface of the first shallow platform structure is not greater than the diameter of the first ball.

[0019] In some embodiments, the second sample processing area is arranged at the output end of the blocking area. The second sample processing area includes a plurality of stepped stacked cavities and a separation liquid cavity. The depths of the plurality of stepped stacked cavities gradually increase from the side close to the blocking area to the side far from the blocking area. The separation liquid cavity is arranged at one end of the stepped stacked cavities far from the first sample processing area.

[0020] In some embodiments, a second exhaust hole is arranged on the stepped stacked cavity close to the first sample processing area, and a separation liquid sampling port is arranged on the separation liquid cavity.

[0021] In some embodiments, the sorted sample area includes:

[0022] A sorted sample cavity, inside which a sample distribution shallow platform structure is arranged. The sample distribution shallow platform structure is communicated with the stepped stacked cavity close to the separation liquid cavity through a second thin tube. One end of the sorted sample cavity is provided with a sheath liquid sampling port;

[0023] A second ball, which is arranged inside the sorted sample cavity;

[0024] A transition platform, which is arranged at one end of the sorted sample cavity far from the sheath liquid sampling port; and

[0025] A deep triangular platform, which is arranged on the side of the transition platform away from the sorted sample cavity.

[0026] In some embodiments, the sample final distribution area is communicated with the deep triangular platform through a distribution thin tube. The sample final distribution area includes a plurality of distribution structures connected to the distribution thin tube. The plurality of distribution structures are arranged around the rotating shaft in a clamped manner. The end of the distribution thin tube is provided with a circular platform structure, and a sample collection structure is arranged circumferentially on the circular platform structure. A third exhaust hole is arranged on the sample collection structure.

[0027] Compared with the prior art, the beneficial effects of the butterfly microfluidic chip provided by the present invention are as follows:

[0028] By providing a shaft clamping position on the chip body, the present invention enables the chip body to be controlled by an external shaft. A whole blood sample diluted to an appropriate multiple is injected into the first sample processing area. Under the action of the shaft, the anticoagulated whole blood sample and the diluent are fully mixed. Then, a separation liquid is injected into the second sample processing area. The sample diluted and mixed well in the first sample processing area, such as the anticoagulated whole blood sample, is stacked on the separation liquid and centrifuged. After centrifugation, the target layer (such as the PBMC layer) can be visually observed. At this time, the target layer is aspirated into the sorting sample area and sheath liquid is added. After the sheath liquid and the sorted sample are evenly mixed, as the shaft drives the chip body to rotate, the liquid is distributed into the final sample distribution area. Thus, the target sample in the final sample distribution area can be extracted, detected, or collected, eliminating the cumbersome steps of manual PBMC extraction, reducing the operation difficulty, and ensuring the purity of PBMC while avoiding the loss of target cells. The final sample distribution area is semicircular and arranged around the shaft clamping position, enabling the sample to be distributed along the pipeline to the final sample distribution area during the rotation of the chip body. The distance between the second sample processing area and the shaft clamping position is greater than the distance between the first sample processing area and the shaft clamping position. Under the action of centrifugal force, the sample is more easily transported from the first sample processing area to the second sample processing area, thereby shortening the extraction time and improving the extraction efficiency of PBMC. Description of the Drawings

[0029] Figure 1 is a schematic structural diagram of the butterfly microfluidic chip described in the present invention.

[0030] Figure 2 is an enlarged schematic diagram of the first sample processing area, the second sample processing area, and the sorting sample area described in the present invention.

[0031] Figure 3 is an enlarged schematic diagram of the blocking area described in the present invention.

[0032] Figure 4 is an enlarged schematic diagram of the final sample distribution area described in the present invention.

[0033] Figure 5 is a schematic diagram of the stratification of the anticoagulated whole blood sample after density gradient centrifugation in the embodiment.

[0034] In the figure, 1 is the chip body; 11 is the shaft clamping position; 12 is the first sample processing area; 121 is the sample mixing and temporary storage cavity; 122 is the sample loading hole; 123 is the first ball; 124 is the first exhaust hole; 125 is the blocking area; 1251 is the convex structure; 1252 is the first shallow platform structure; 1253 is the wax valve structure; 1254 is the deep platform structure; 1255 is the second shallow platform structure; 13 is the second sample processing area; 131 is the first stepped stacked cavity; 132 is the second exhaust hole; 133 is the second stepped stacked cavity; 134 is the third stepped stacked cavity; 135 is the fourth stepped stacked cavity; 136 is the fifth stepped stacked cavity; 137 is the separation liquid loading port; 138 is the separation liquid cavity; 14 is the sorted sample area; 141 is the sorted sample cavity; 142 is the sample distribution shallow platform structure; 143 is the sheath fluid injection port; 144 is the second ball; 145 is the transition platform; 146 is the deep triangular platform; 15 is the final sample distribution area; 151 is the distribution structure; 152 is the round platform structure; 153 is the sample collection structure; 154 is the third exhaust hole; 16 is the first thin tube; 17 is the second thin tube; 18 is the distribution thin tube;

[0035] 2. Red blood cell and sediment layer;

[0036] 3. Separation liquid layer;

[0037] 4. PBMC layer;

[0038] 5. Serum and small molecule layer. Specific embodiments

[0039] The following combines the accompanying drawings and embodiments to further describe in detail the specific embodiments of the present invention. The following embodiments are used to illustrate the present invention, but are not used to limit the scope of the present invention.

[0040] In the description of the present invention, it should be understood that the orientation or positional relationships indicated by the terms "close to", "far from", "center of the circle", "inside (side)", "inner wall", "outside (side)", "side end", "between", "around", "input end", "output end", "left side", "right side", etc. in the present invention are based on the orientation or positional relationships shown in the accompanying drawings, and are 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 of the present invention.

[0041] In addition, it should be understood that the terms "deep" and "shallow" used in the present invention are only for the convenience of distinguishing the structures referred to in the present invention, rather than being used to limit the actual "depth" of the structures referred to, and therefore should not be construed as a limitation of the present invention.

[0042] The terms "first", "second", "third", etc. are only for descriptive purposes and should not be construed as indicating or implying relative importance. In addition, unless otherwise clearly specified and defined, the terms "installed", "connected", and "joined" 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 a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the communication inside two components. 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 circumstances.

[0043] To facilitate the description of the working principle of the present invention, the following explanations are made: It is required that the rotating shaft adapted to the rotating shaft clamping position 11 can achieve the functions of low speed, high speed, and left - right shaking. In addition, the position of the holes on the surface of the butterfly - type microfluidic chip corresponds to the position after film sealing. Here, film sealing refers to using a single - sided pressure - sensitive adhesive for pipeline sealing. After punching holes at the corresponding positions of the sample inlet and the exhaust hole, they should be closely attached to the butterfly - type microfluidic chip. The advantage of using a single - sided pressure - sensitive adhesive is that there is no or very little adhesiveness at the positions where the pressure - sensitive adhesive is not stressed, meeting the requirements of chip pipeline sealing in CNC processing. When the film sealing, such as the adhesion of the pressure - sensitive adhesive to the microfluidic chip, requires an increase in rotational speed, the movement of the ball, and the application of an external sheath fluid (and pressure), there are no defects such as liquid leakage or bursting.

[0044] As Figure 1 As shown, the present invention provides a butterfly - type microfluidic chip, which includes a chip body 1. A rotating shaft clamping position 11 is provided at the center of the circle of the chip body 1. Among them, the provided rotating shaft clamping position 11 can be adapted to an external rotating shaft and can stably hold it without shaking, avoiding damage to the butterfly - type microfluidic chip and preventing the reduction of the purity of the extracted PBMC or the inability to extract PBMC. To further ensure the stability and balance of the rotation of the chip body 1, it is preferred that the chip body 1 is a perfect circle.

[0045] Please refer to Figure 1-2, in a specific example of the present invention, four major regions are provided on the chip body 1, namely a first sample processing region 12, a second sample processing region 13, a sorted sample region 14, and a sample final distribution region 15. Among them, the first sample processing region 12 is connected to the second sample processing region 13 through a first capillary 16, the second sample processing region 13 is connected to the sorted sample region 14 through a second capillary 17, and the sorted sample region 14 is connected to the sample final distribution region 15 through a distribution capillary 18. Through the first capillary 16, the second capillary 17, and the distribution capillary 18, it is convenient for the fluid medium to flow into the second sample processing region 13, the sorted sample region 14, and the sample final distribution region 15 in sequence through the first sample processing region 12, avoiding manual intervention and improving the extraction efficiency. Specifically: Inject the whole blood sample diluted by an appropriate multiple into the first sample processing region 12. Under the action of the rotating shaft, the anticoagulated whole blood sample and the diluent are fully mixed. Inject the separation liquid into the second sample processing region 13, stack the diluted and mixed sample in the first sample processing region 12, such as the anticoagulated whole blood sample, on top of the separation liquid and perform centrifugation. After centrifugation, the target layer (such as the PBMC layer, see Figure 5 ) can be seen with the naked eye. At this time, the target layer is aspirated into the sorted sample region 14 and sheath fluid is added. After the sheath fluid and the sorted sample are mixed evenly, as the rotating shaft drives the chip body 1 to rotate, the liquid is distributed into the sample final distribution region 15, so that the target sample in the sample final distribution region 15 can be extracted, detected, or collected, saving the cumbersome steps of manually extracting PBMC, reducing the operation difficulty, and at the same time ensuring the purity of PBMC and avoiding the loss of target cells; the sample final distribution region 15 is semicircular and arranged around the rotating shaft clamping position 11, so that the sample can be distributed along the pipeline to the sample final distribution region 15 during the rotation of the chip body 1. The distance between the second sample processing region 13 and the rotating shaft clamping position 11 is greater than the distance between the first sample processing region 12 and the rotating shaft clamping position 11. Under the action of centrifugal force, it is beneficial to accelerate the speed of the sample from the first sample processing region 12 to the second sample processing region 13, shorten the extraction time, and improve the extraction efficiency of PBMC.

[0046] In some examples of the present invention, as shown in Figure 2 , the first sample processing region 12 includes a sample mixing and temporary storage cavity 121 and a first ball 123. The first ball 123 is arranged inside the sample mixing and temporary storage cavity 121 and always moves along the inner wall of the sample mixing and temporary storage cavity 121 away from the center of the chip body 1. Among them, a sampling hole 122 is opened at one end of the sample mixing and temporary storage cavity 121 close to the center of the chip body 1. After mixing the sample, such as undiluted anticoagulated whole blood and the diluent evenly at a ratio of 1:1, the sample is added into the sample mixing and temporary storage cavity 121 from the position of the sampling hole 122.

[0047] In some examples of the present invention, it is required that the size of the sample loading hole 122 can meet the requirement that most pipettes can load samples without leakage, and the sample can be easily loaded. After the sample is loaded, the sample mixing and temporary storage cavity 121 needs to be sealed. Sealing the sample mixing and temporary storage cavity 121 means sealing the sample loading hole 122 with a sticker or other items to prevent the sample from flowing back during the rotation of the chip body 1.

[0048] Specifically, a sample such as a whole blood sample diluted by one time enters the sample mixing and temporary storage cavity 121. The rotating shaft drives the chip body 1 to stably shake left and right, and the first ball 123 located in the sample mixing and temporary storage cavity 121 shakes left and right accordingly, so as to mix the sample such as diluted blood.

[0049] In other examples of the present invention, the diluent is physiological saline or PBS or other solution for dilution.

[0050] In one embodiment, the first ball 123 is a small ball with a size less than 1 / 3 of the depth of the sample mixing and temporary storage cavity 121, and is placed in the sample mixing and temporary storage cavity 121 before the chip body 1 is sealed with a film to ensure that it is not adhered to the pressure-sensitive adhesive, that is, it can roll in the sample mixing and temporary storage cavity 121 after the film is sealed, so as to achieve the effect of stirring and mixing the sample and the diluent.

[0051] In one embodiment, a first exhaust hole 124 is further provided at one end of the sample mixing and temporary storage cavity 121 close to the center of the chip body 1. A sample such as a whole blood sample diluted by one time enters the sample mixing and temporary storage cavity 121. The rotating shaft drives the chip body 1 to stably shake left and right. At this time, it is required that the rotation speed of the rotating shaft can make the liquid move along the inner wall away from the center of the circle end, and will not flow out from the position of the first exhaust hole 124. The first exhaust hole 124 is used to balance the internal and external air pressures of the sample mixing and temporary storage cavity 121 to avoid the pressure-sensitive adhesive from bursting after the film is sealed.

[0052] In one embodiment, a blocking area 125 is provided at one end of the sample mixing and temporary storage cavity 121 far from the center of the chip body 1. The blocking area 125 and the sample loading hole 122 are diagonally arranged on the sample mixing and temporary storage cavity 121, so that the sample loading and the sample discharging are at different positions. When the rotating shaft drives the chip body 1, the mixed sample can flow out from the blocking area 125 more easily, avoiding the low liquid output of the mixed sample and resulting in the inability to improve the purity of PBMC.

[0053] In one embodiment, the first exhaust hole 124 and the blocking area 125 are arranged on the same side. To ensure that the rotation speed of the rotating shaft can make the liquid move along the inner wall away from the center of the circle end and will not flow out from the first exhaust hole 124, for this reason, the distance between the first exhaust hole 124 and the center of the chip body 1 needs to be set to be less than the distance between the blocking area 125 and the center of the chip body 1.

[0054] In the specific examples of the present invention, please refer to Figure 3, the blocking area 125 includes a convex structure 1251, a first shallow platform structure 1252, a wax valve structure 1253, a deep platform structure 1254, and a second shallow platform structure 1255. Among them, the convex structure 1251 is provided on the inner wall of the sample mixing and temporary storage cavity 121 away from the center of the chip body 1. The convex structure 1251 is arranged in a triangular structure, which helps the ball 10 to move back and forth in the sample mixing and temporary storage cavity 121 to achieve the purpose of balancing the sample and the diluent.

[0055] In one embodiment, the first shallow platform structure 1252 is provided in the sample mixing and temporary storage cavity 121 and on one side of the convex structure 1251. To prevent the first ball 123 from entering the first shallow platform structure 1252 during shaking and causing blockage, the depth of the first shallow platform structure 1252 is designed to be less than the depth of the sample mixing and temporary storage cavity 121. Due to the height difference between the first shallow platform structure 1252 and the sample mixing and temporary storage cavity 121, it can play a good blocking role for the first ball 123. To further improve the blocking effect on the first ball 123, the depth of the first shallow platform structure 1252 is designed to be greater than the height of the convex structure 1251, so that the distance between the bottom surface of the convex structure 1251 and the bottom surface of the first shallow platform structure 1252 is not greater than the diameter of the first ball 123. At this time, it can better limit the first ball 123 from straying into the first shallow platform structure 1252.

[0056] Please continue to combine Figure 3 , in one embodiment, the wax valve structure 1253 is provided outside the sample mixing and temporary storage cavity 121 and corresponds to the first shallow platform structure 1252, and the deep platform structure 1254 is provided on the side of the wax valve structure 1253 away from the first shallow platform structure 1252. Preferably, the wax valve structure 1253 is a cuboid hard wax block, which is embedded in the pipeline structure of the chip body 1 to form a channel blocking the connection between the sample mixing and temporary storage cavity 121 and the second sample processing area 13, avoiding the sample and the diluent from entering the second sample processing area 13 before being mixed and affecting the extraction purity of PBMC.

[0057] In one embodiment, the wax valve structure 1253 does not change its surface at room temperature (10°C - 25°C). As the external temperature slowly rises to 30°C, it begins to melt. As the temperature rises (not exceeding 35°C), the wax valve structure 1253 shows obvious changes. When the chip body 1 is shaken left and right by the rotating shaft, the mixed sample moves towards the wax valve structure 1253, passes through the wax valve structure 1253, enters the first shallow platform structure 1252, and then enters the deep platform structure 1254.

[0058] In one embodiment, the depth of the deep platform structure 1254 is greater than the depths of the first shallow platform structure 1252 and the second shallow platform structure 1255. When a sample, such as anticoagulated whole blood diluted by one time, enters the deep platform structure 1254, the rotating shaft rotates counterclockwise. Since the rotating shaft drives the chip body 1 to rotate in a single direction, the melted wax in the wax valve structure 1253 will first enter the deep platform structure 1254 and fill the bottom, causing the bottom of the deep platform structure 1254 to gradually rise, and its depth is equal to or slightly lower than the depth of the first shallow platform structure 1252 or the second shallow platform structure 1255, so that the sample can flow into the second shallow platform structure 1255 after passing through the deep platform structure 1254.

[0059] In one embodiment, the second shallow platform structure 1255 is provided with a first thin tube 16 for guiding the sample mixing medium to the second sample processing area 13. As the centrifugal force increases, the rotational speed ranges are tested at 500 rpm / min, 1000 rpm / min, 1500 rpm / min, 3000 rpm / min, and 5000 rpm / min. The test shows that when the speed is medium, the melted wax can spread flat on the bottom of the deep platform structure 1254 and does not enter the second sample processing area 13 through the first thin tube 16.

[0060] As Figure 2 , in some examples of the present invention, the second sample processing area 13 is provided at the output end of the blocking area 125, that is, the liquid outlet end of the first thin tube 16 is connected to the second sample processing area 13. Specifically, the second sample processing area 13 includes a plurality of stepped stacked cavities and a separation liquid cavity 138. The depths of the plurality of stepped stacked cavities gradually increase from the side close to the blocking area 125 to the side far from the blocking area 125, and the separation liquid cavity 138 is provided at one end of the stepped stacked cavities far from the first sample processing area 12.

[0061] To further explain the principle of the present invention, please combine Figure 2 and Figure 5 , and now define the plurality of stepped stacked cavities. The plurality of stepped stacked cavities sequentially include a first stepped stacked cavity 131, a second stepped stacked cavity 133, a third stepped stacked cavity 134, a fourth stepped stacked cavity 135, and a fifth stepped stacked cavity 136 from the side far from the separation liquid cavity 138 to the side close to the separation liquid cavity 138. Among them, the depth of the first stepped stacked cavity 131 is less than that of the second stepped stacked cavity 133, which is less than that of the third stepped stacked cavity 134, which is less than that of the fourth stepped stacked cavity 135, which is less than that of the fifth stepped stacked cavity 136, thus forming a stepped stacked state. The purpose is to enable a sample, such as a whole blood sample diluted by one time and with a uniform medium, to diffuse away from the center and slowly and evenly stack on the separation liquid at a low rotational speed or when stopped rotating.

[0062] In one embodiment, a second exhaust hole 132 is provided on the stepped stacked cavity near the first sample processing area 12 for balancing the internal and external air pressures of the second sample processing area 13, and a separation liquid adding port 137 is provided on the separation liquid cavity 138 for adding separation liquid into the separation liquid cavity 138.

[0063] In one embodiment, the separation liquid is a mixed liquid of Ficoll, hydroxyethyl starch 550 and meglumine diatrizoate, or a separation liquid mainly composed of dextran (dextrin) and meglumine diatrizoate.

[0064] In another embodiment, the principle of action of the separation liquid is as follows: Mononuclear cells in peripheral blood include lymphocytes, monocytes, etc., and their volume, morphology and density are different from those of other cells. The density of red blood cells and granulocytes in blood is relatively large, about 1.090 g / ml, but the density of lymphocytes and monocytes is 1.075 - 1.090 g / ml, and that of platelets is 1.030 - 1.035 g / ml. After lamination treatment by appropriate means, with the action of centrifugation and the separation liquid, cells with relatively large density such as red blood cells and granulocytes will settle at the bottom. At this time, white blood cells are still in the upper layer of the separation liquid and in the separation liquid layer (a small number), and substances with the smallest density such as platelets are in the uppermost layer.

[0065] In some preferred solutions, the diluted sample such as anticoagulated whole blood is gently stacked on the separation liquid, and centrifuged without mixing or shaking. After centrifugation, the target layer such as the PBMC layer can be seen with the naked eye. The function of the stepped stacked cavity is to simulate the manual gentle stacking process. After the sample such as anticoagulated whole blood diluted by one time slowly flows from the stepped layer to the separation liquid cavity 138 and the rotating shaft rotates for centrifugation, layering occurs; as Figure 5 shown, from top to bottom are the serum small molecule layer 5, the PBMC layer 4, the separation liquid layer 3, and the red blood cell and sediment layer 2.

[0066] In some embodiments, the separation liquid is added in advance from the separation liquid adding port 137 and then sealed with a sticker or other items. It is required that the separation liquid be added before the wax valve is melted by temperature / before the sample such as anticoagulated whole blood diluted by one time enters the stepped stacked cavity.

[0067] In some embodiments, the depth of the first capillary 16 is the same as that of the second capillary 17, and is the same as the depth of the first shallow platform structure 1252 or the second shallow platform structure 1255.

[0068] In some embodiments, the width of the first capillary 16 is 2 times wider than that of the second capillary 17. The reason is that it can avoid the risk of the wax flowing into the stepped stacked cavity after the wax in the wax valve structure melts, and can prevent the first capillary 16 from being blocked and avoid chip scrapping.

[0069] In some embodiments, a necessary condition for lamination is that the rotating shaft rotates at a certain speed. Through experiments, it is preferably rotated at 1900 rpm / min for 5 minutes to achieve a better lamination effect.

[0070] In some embodiments, the second thin tube 17 is provided on the left side of the middle part of the stepped lamination cavity. The reason is that after the sample, such as anticoagulated whole blood diluted by one time, undergoes density gradient centrifugation, the PBMC layer is roughly located in the second stepped lamination cavity 133 and the third stepped lamination cavity 134. After the sample lamination is completed and stands still, due to the influence of inertial force, the liquid will be sucked into the sorting sample area 14 by the second thin tube 17. That is, the PBMC layer 4, the separation liquid layer 3, etc. will flow into the sorting sample area 14 along the second thin tube 17.

[0071] In some embodiments, after centrifugal lamination and standing still, seal the second exhaust hole 132 with other items such as stickers or tapes to prevent air leakage; after the liquid is transferred to the sorting sample area 14 by the second thin tube 17, then seal the first exhaust hole 124 in the sample mixing and temporary storage cavity 121 with other items such as stickers or tapes to prevent air leakage.

[0072] In other examples, the second exhaust hole 132 is lifted upward and forms an acute-angled corner when it falls, so as to prevent backflow when perfusion sheath fluid into the sorting sample area 14 after standing still.

[0073] The volume of the above cavity is adapted to the sample loading volume and the lamination and sorting volume. The volume adaptation means that when designing the total volume of the cavity, volume adjustment is carried out according to the sample loading amount, dilution factor, etc. The vast majority of the adjustment refers to improvement in the depth of the cavity, not the width.

[0074] Please continue to refer to Figure 2 , in a specific example of the present invention, the sorting sample area 14 includes a sorting sample cavity 141, a second ball 144, a transition platform 145, and a deep triangular platform 146. Among them, a sample distribution shallow platform structure 142 is provided inside the sorting sample cavity 141. The liquid outlet end of the second thin tube 17 communicates with the shallow platform structure 142. The depth of the shallow platform structure 142 is shallower than the depth of the sorting sample cavity 141. When introducing the target layer liquid into the sorting sample cavity 141, it can also play a role in preventing backflow.

[0075] In some embodiments, a sheath fluid injection port 143 is opened at one end of the sorting sample cavity 141. The sheath fluid injection port 143 requires an external associated syringe to provide the driving force for the sheath fluid to flow; in specific operations, first install a metal such as a stainless steel needle or a flat head syringe needle slightly larger than its circumference in the sheath fluid injection port 143 and connect it with hot melt glue; then connect the metal joint installed in the sheath fluid injection port 143 with a hard rubber tube to a syringe. The syringe contains sheath fluid such as PBS, physiological saline, or other liquid media.

[0076] In some embodiments, the syringe needs to be clamped on the injection pump for operation to ensure that the syringe can be firmly clamped and the flow rate can be adjusted. Preferably, since the control of the flow rate has a certain impact on the subsequent distribution and sorting effect, when the injection pump drives the syringe to inject the sheath fluid at a flow rate of 200 μl - 1000 μl / min, the impact is relatively small.

[0077] Refer to Figure 2 and Figure 4 , in some examples of the present invention, the sample final distribution area 15 is communicated with the deep triangular platform 146 through the distribution capillary 18. The sample final distribution area 15 includes a plurality of distribution structures 151 connected to the distribution capillary 18. The plurality of distribution structures 151 are arranged around the rotating shaft clamping position 11. A third exhaust hole 154 is provided on the sample collection structure 153.

[0078] In some embodiments, the sheath fluid inlet 143 is always closed until the second capillary 17 sucks the PBMC layer in the stepped stacked cavity into the sorting sample cavity 141 completely, and the first exhaust hole 124 and the second exhaust hole 132 in the sample mixing and temporary storage cavity 121 are closed. Then, the sheath fluid is slowly added. When the sheath fluid is injected, the third exhaust hole 154 is always in an open state.

[0079] In some embodiments, the second ball 144 is arranged inside the sorting sample cavity 141 and is used to mix the sheath fluid and the sorted sample evenly. As the rotating shaft drives the chip body 1 to rotate, the liquid is distributed into the transition platform 145. The use of the second ball 144 is the same as that of the first ball 123, and it needs to be driven by the rotating shaft to rotate left and right. The difference from the first ball 123 is that the second ball 144 does not need to mix the sheath fluid and the sorted sample absolutely evenly, and only plays a slight mixing role.

[0080] In some embodiments, the transition platform 145 is arranged at one end of the sorting sample cavity 141 away from the sheath fluid inlet 143, and the deep triangular platform 146 is arranged on the side of the transition platform 145 away from the sorting sample cavity 141. Among them, the depth of the transition platform 145 is the same as that of the sorting sample cavity 141 and is used as a buffer area for the sheath fluid and the sorted sample. The depth of the deep triangular platform 146 is deeper than that of the transition platform 145, and its function is to connect the deeper distribution capillary 18 to facilitate the distribution of the sample into each distribution structure 151.

[0081] In some embodiments, the sheath fluid serves to provide external power to the sorted sample mixture and further distribute the liquid obtained in the sample sorting cavity; the sample is driven by the pressure of an external injection pump to flow into the sheath fluid inlet 143 with the sheath fluid, reach the transition platform 145, and then enter the deep triangular platform 146 due to the action of the flow direction of the sheath fluid and centrifugal force; thereafter, the chip stops rotating with the rotating shaft and is in a stationary state.

[0082] In other examples, the chip can be placed on a horizontal plane. After connecting the syringe filled with the sheath fluid, the subsequent power only comes from the sheath fluid.

[0083] The above cavity volume is three times or more of the original sample volume, which serves to ensure that there is sufficient space for sheath fluid injection and prevent the sealing film from bursting when the injection speed of the sheath fluid is relatively high.

[0084] The sorted sample and the sheath fluid mixture flow into the respective distribution structures 151 along the distribution capillary 18 as the sheath fluid flows into the sheath fluid inlet. The distribution capillary 18 is set in a semi-circular structure, so that the liquid, such as the mixture of the sorted sample and the sheath fluid, fills the first distribution structure 151 first and then enters the second distribution structure 151 for filling, and so on, to avoid affecting subsequent extraction, detection or collection due to uneven distribution, thereby improving the purity of PBMC.

[0085] In other examples, a circular platform structure 152 is provided at the end of the distribution capillary 18, and a sample collection structure 153 is provided circumferentially on the circular platform structure 152. As time goes by and the sheath fluid is continuously added, the sorted sample is continuously diluted, and the liquid volume rises and enters the circular platform structure 152 and the sample collection structure 153. The circular platform structure 152 can play a role in preventing backflow, while the sample collection structure 153 can be used as a waste liquid temporary storage or as a final reaction area.

[0086] In other examples, the amount of sheath fluid added is related to the total volume of each distribution cavity and the total volume of the circular sample collection structure, and it is required not to exceed the maximum capacity to avoid the bursting of the sealing film or the liquid flowing out of the third exhaust hole 154 due to the addition of the sheath fluid;

[0087] In other examples, the depth of the third exhaust hole 154 should be set relatively shallow to avoid affecting the normal function of the sample collection structure 153.

[0088] In other examples, a metal needle is embedded in the sheath fluid inlet 143, and corresponding measures are also taken at the symmetric position along the center of the circle to balance the centrifugal force at both ends.

[0089] The working process of the present invention is as follows:

[0090] 1. Inject a sample, such as a diluted whole blood sample (diluted by a factor of two), into the sample mixing and temporary storage chamber 121. The rotating shaft drives the chip body 1 to shake, and the first ball 123 located in the sample mixing and temporary storage chamber 121 shakes accordingly, thereby mixing the sample, such as the diluted whole blood sample. After adding the sample, open the first exhaust hole 124 and seal the sample loading hole 122.

[0091] 2. The melted wax in the wax valve structure 1253 will first enter the deep platform structure 1254 and fill the bottom. The sample flows through the first shallow platform structure 1252 and the deep platform structure 1254 and then into the second shallow platform structure 1255, and then enters the first stepped stacked cavity 131 through the first thin tube 16. Before the wax valve is melted by the temperature / before the sample, such as diluted anticoagulated whole blood, enters the stepped stacked cavity, add separation liquid from the separation liquid sample loading port 137 first, seal the separation liquid sample loading port 137 and open the second exhaust hole 132.

[0092] 3. The sample, such as diluted anticoagulated whole blood, enters the stepped stacked cavity for centrifugal stratification. After the centrifugal stratification stops, seal the second exhaust hole 132 to prevent air leakage. After the target layer, such as the PBMC layer, is transferred to the sorting sample area 14 through the second thin tube 17, seal the first exhaust hole 124.

[0093] 4. Aspirate all the PBMC layers in the stepped stacked cavity into the sorting sample cavity 141, mix them with the second ball 144, and slowly add sheath liquid with the first exhaust hole 124 and the second exhaust hole 132 closed. The third exhaust hole 154 is always open. The sample is driven by the pressure of an external injection pump to flow the sheath liquid from the sheath liquid sample loading port 143 into the transition platform 145, and then enters the deep triangular platform 146 due to the flow direction of the sheath liquid and the centrifugal force.

[0094] 5. The mixed solution of the sorted sample and the sheath liquid flows into each distribution structure 151 along the distribution thin tube 18 with the inflow of the sheath liquid from the sheath liquid sample loading port. Among them, the distribution thin tube 18 is set in a semicircular structure, so that the liquid, such as the mixed solution of the sorted sample and the sheath liquid, can fill the first distribution structure 151 first, and then enter the second distribution structure 151 for filling, and finally reach the circular platform structure 152 and the sample collection structure 153.

[0095] In summary, for the butterfly - type microfluidic chip provided by the present invention, after the whole blood sample is injected into the first sample processing area 12, the target sample can be extracted, detected or collected in the sample final distribution area 15, which omits the cumbersome steps of manual extraction of PBMC, reduces the operation difficulty, ensures the purity of PBMC, avoids the loss of target cells, and can shorten the extraction time and improve the extraction efficiency of PBMC.

[0096] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the technical principle of the present invention, several improvements and replacements can be made, and these improvements and replacements should also be regarded as the protection scope of the present invention. The basic principles, main features and advantages of the present invention have been shown and described above. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above preferred embodiments. The embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention.

[0097] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in the embodiments can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A butterfly microfluidic chip, characterized in that, It includes a chip body, a rotating shaft clamping position is provided at the center of the circle of the chip body, and a first sample processing area, a second sample processing area, a sorted sample area, and a final sample distribution area are respectively arranged on the chip body; wherein, the first sample processing area is connected to the second sample processing area through a pipeline, the second sample processing area is connected to the sorted sample area through a pipeline, the sorted sample area is connected to the final sample distribution area through a pipeline, the final sample distribution area is semicircular and arranged around the rotating shaft clamping position, and the distance between the second sample processing area and the rotating shaft clamping position is greater than the distance between the first sample processing area and the rotating shaft clamping position; The first sample processing area includes: A sample mixing and temporary storage cavity, a sampling hole is provided at one end of the sample mixing and temporary storage cavity close to the center of the circle of the chip body, a blocking area is provided at the end of the sample mixing and temporary storage cavity far from the center of the circle of the chip body, and the blocking area and the sampling hole are diagonally arranged on the sample mixing and temporary storage cavity; and A first ball, the first ball is arranged inside the sample mixing and temporary storage cavity and always moves along the inner wall of the sample mixing and temporary storage cavity at the end far from the center of the circle of the chip body; The blocking area includes: A convex structure, the convex structure is arranged on the inner wall of the sample mixing and temporary storage cavity far from the center of the circle of the chip body; A first shallow platform structure, the first shallow platform structure is arranged inside the sample mixing and temporary storage cavity and on one side of the convex structure; A wax valve structure, the wax valve structure is arranged outside the sample mixing and temporary storage cavity and corresponds to the first shallow platform structure; A deep platform structure, the deep platform structure is arranged on the side of the wax valve structure far from the first shallow platform structure; and A second shallow platform structure, the second shallow platform structure is arranged corresponding to the deep platform structure, and the second shallow platform structure is provided with a first thin tube for guiding the sample mixing medium to the second sample processing area; The depth of the first shallow platform structure is less than the depth of the sample mixing and temporary storage cavity, and the depth of the first shallow platform structure is less than the height of the convex structure, and the distance between the bottom surface of the convex structure and the bottom surface of the first shallow platform structure is not greater than the diameter of the first ball.

2. The butterfly microfluidic chip according to claim 1, wherein A first exhaust hole is also provided on the sample mixing and temporary storage cavity, the first exhaust hole and the blocking area are on the same side, and the distance between the first exhaust hole and the center of the circle of the chip body is less than the distance between the blocking area and the center of the circle of the chip body.

3. A butterfly microfluidic chip according to claim 1, characterized in that, The diameter of the first ball is less than one-third of the depth of the sample mixing and temporary storage cavity.

4. A butterfly microfluidic chip according to claim 1, characterized in that, The second sample processing area is arranged at the output end of the blocking area, the second sample processing area includes a plurality of stepped stacked cavities and a separation liquid cavity, the depths of the plurality of stepped stacked cavities gradually increase from the side close to the blocking area to the side far from the blocking area, and the separation liquid cavity is arranged at one end of the stepped stacked cavities far from the first sample processing area.

5. A butterfly microfluidic chip according to claim 4, wherein A second exhaust hole is provided on the stepped stacked cavity close to the first sample processing area, and a separation liquid sampling port is provided on the separation liquid cavity.

6. A butterfly microfluidic chip according to claim 4, characterized in that, The sorted sample area includes: Sorting sample cavity, a sample distribution shallow platform structure is provided inside the sorting sample cavity, the sample distribution shallow platform structure is communicated with a stepped laminated cavity close to the separation liquid cavity through a second thin tube, and a sheath liquid injection port is opened at one end of the sorting sample cavity; Second ball, the second ball is arranged inside the sorting sample cavity; Transition platform, the transition platform is arranged at one end of the sorting sample cavity away from the sheath liquid injection port; and Deep triangular platform, the deep triangular platform is arranged on one side of the transition platform away from the sorting sample cavity.

7. A butterfly microfluidic chip according to claim 6, wherein, The final sample distribution area is communicated with the deep triangular platform through a distribution thin tube, the final sample distribution area includes a plurality of distribution structures communicated with the distribution thin tube, the plurality of the plurality of distribution structures are arranged around the rotation axis in a clamping manner, a positive circular platform structure is arranged at the end of the distribution thin tube, a sample collection structure is arranged circumferentially on the positive circular platform structure, and a third exhaust hole is opened on the sample collection structure.

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