CN115814870A - Butterfly type micro-fluidic chip - Google Patents

Abstract

The invention relates to the technical field of medical instruments, and particularly discloses a butterfly type microfluidic chip which comprises a chip body, wherein a rotating shaft clamping position is arranged at the center of the chip body, and a first sample processing area, a second sample processing area, a sorting sample area and a final sample distribution area are respectively arranged on the chip body; wherein the first sample processing region is piped to the second sample processing region, the second sample processing region is piped to the sort sample region, the sort sample region is piped to the sample final distribution region, the sample final distribution region is semi-circular and is arranged around the spindle detent, and the distance between the second sample processing region and the spindle detent is greater than the distance between the first sample processing region and the spindle detent. The method saves the complicated steps of manually extracting the PBMC, reduces the operation difficulty, ensures the purity of the PBMC, avoids the loss of target cells, and can improve the extraction efficiency of the PBMC.

Description

Butterfly type micro-fluidic chip

Technical Field

The invention relates to the technical field of medical instruments, in particular to a butterfly type micro-fluidic chip.

Background

PBMC (peripheral blood mononuclear cell) mainly comprises cells with mononuclear cells in blood, mainly including lymphocytes (T/B), monocytes, phagocytes, dendritic cells and other small cell types. Because of the huge number of red blood cells and platelets in whole blood, the existence of these cells has great influence on antibody labeling and flow analysis, and thus it is likely to obtain wrong results, which results in that when whole blood is directly used to detect lymphocytes or monocytes clinically, the obtained experimental results are generally not approved, while each subgroup of lymphocytes keeps a certain proportion and quantity in normal organisms, and it is clinically found that when the proportion is out of regulation, the immune function in vivo may be disordered, thereby causing diseases. By carrying out immune typing (qualitative) and quantitative analysis on lymphocytes in blood, the collective immune function can be more accurately evaluated, and the method has important significance for clinically diagnosing immunodeficiency diseases and autoimmune diseases and guiding immunotherapy. Therefore, in the laboratory, the whole blood PBMCs are usually extracted to simulate the in vitro blood immune environment for experiment, so that mononuclear cells are extracted for detection.

The mononuclear cells in the peripheral blood include lymphocytes, monocytes, and the like, and have a volume, a shape, and a density different from those of other cells. The density of erythrocytes and granulocytes in blood is large, about 1.090g/ml, but the density of lymphocytes and monocytes is 1.075-1.090 g/ml, and the density of platelets is 1.030-1.035 g/ml. After the stacking process by suitable means, the denser cells such as erythrocytes and granulocytes will settle to the bottom by the action of the centrifugation and separation liquid. In this case, leukocytes remain on the upper layer and the separation liquid layer (few number), and the material such as platelets having the lowest density is on the uppermost layer. And finally, sucking out the PBMC layer through a pipette gun to obtain a PBMC layer mixed solution for a laboratory.

The PBMC is extracted by manually sucking out the PBMC through observing the position of a PBMC layer by naked eyes after density gradient centrifugation at present, the purity of the PBMC is low, the operation has high requirement on training, the operation needs to be repeatedly practiced, and the popularization of the method is not facilitated; moreover, the PBMC component extracted is complex, and downstream analysis also requires operations such as washing, etc., and the probability of target cell loss is increased by removing the separation liquid or other unnecessary media.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: the defects of low purity and complex operation of artificially extracting the target cell PBMC are avoided.

In order to solve the technical problems, the invention provides a butterfly-type microfluidic chip, which comprises a chip body, wherein a rotating shaft clamp is arranged at the center of a circle of the chip body, a first sample processing area, a second sample processing area, a sorting sample area and a sample final distribution area are respectively arranged on the chip body, wherein the first sample processing area is connected with the second sample processing area through a pipeline, the second sample processing area is connected with the sorting sample area through a pipeline, the sorting sample area is connected with the sample final distribution area through a pipeline, the sample final distribution area is semicircular and is arranged around the rotating shaft clamp, and the distance between the second sample processing area and the rotating shaft clamp is greater than the distance between the first sample processing area and the rotating shaft clamp.

In some of these embodiments, the first sample processing region comprises:

a sample mixing temporary storage cavity, wherein a sample loading hole is formed in one end, close to the circle center of the chip body, of the sample mixing temporary storage cavity, a blocking area is formed in one end, far away from the circle center of the chip body, of the sample mixing temporary storage cavity, and the blocking area and the sample loading hole are arranged on the sample mixing temporary storage cavity in a diagonal manner; and

the first ball is arranged in the sample mixing temporary storage cavity and always follows the sample mixing temporary storage cavity to be kept away from the inner wall of one end of the circle center of the chip body.

In some embodiments, the sample mixing temporary storage cavity is further provided with a first vent hole, the first vent hole and the blocking area are arranged on the same side, and the distance between the first vent hole and the center of the chip body is smaller than the distance between the blocking area and the center of the chip body.

In some embodiments, the diameter of the first ball bearing is less than one third of the depth of the sample mixing buffer chamber.

In some of these embodiments, the blocking region comprises:

the protruding structure is arranged on the inner wall of the sample blending temporary storage cavity, which is far away from the inner wall of the circle center of the chip body;

the first shallow platform structure is arranged in the sample blending temporary storage cavity and is arranged on one side of the bulge structure;

the wax valve structure is arranged on the outer side of the sample blending temporary storage cavity and corresponds to the first shallow platform structure;

the deep platform structure is arranged on one side, away from the first shallow platform structure, of the wax valve structure; and

the second shallow platform structure is arranged corresponding to the deep platform structure, and is provided with a first thin pipe for guiding a sample mixing medium to the second sample processing area.

In some embodiments, the depth of the first shallow platform structure is less than the depth of the sample mixing temporary storage cavity, and the depth of the first shallow platform structure is greater than the height of the raised structure, the distance between the bottom surface of the protruding structure and the bottom surface of the first shallow platform structure is not larger than the diameter of the first ball.

In some embodiments, the second sample processing region is disposed at an output end of the blocking region, the second sample processing region includes a plurality of step-stacked cavities and a separation liquid cavity, depths of the step-stacked cavities gradually increase from a position close to the blocking region to a position far away from the blocking region, and the separation liquid cavity is disposed at an end of the step-stacked cavity far away from the first sample processing region.

In some embodiments, a second vent hole is disposed on the stepped lamination cavity near the first sample processing region, and a separation solution sample port is disposed on the separation solution cavity.

In some of these embodiments, the sort sample region comprises:

the separation device comprises a separation sample cavity, wherein a sample distribution shallow platform structure is arranged on the inner side of the separation sample cavity and communicated with a stepped laminated cavity close to one side of the separation liquid cavity through a second thin tube, and one end of the separation sample cavity is provided with a sheath liquid injection port;

the second ball is arranged on the inner side of the sorting sample cavity;

the transition platform is arranged at one end, far away from the sheath fluid injection port, of the sorted sample cavity; and

the deep triangular platform is arranged on one side, far away from the sorting sample cavity, of the transition platform.

In some of these embodiments, the final sample distribution area with dark triangle platform passes through the distribution tubule intercommunication, the final sample distribution area include a plurality of with the distribution structure that the distribution tubule is linked together, a plurality of distribution structure center on the setting of pivot screens, the end of distribution tubule is provided with the perfect circle platform structure, the circumference of perfect circle platform structure is provided with the sample collection structure, the structural third exhaust hole of having seted up of sample collection.

Compared with the prior art, the butterfly type micro-fluidic chip provided by the invention has the beneficial effects that:

the chip body is provided with the rotating shaft clamp position, so that the chip body can be controlled by an external rotating shaft, a whole blood sample diluted by a proper multiple is injected into the first sample processing area, an anticoagulated whole blood sample and a diluent are fully and uniformly mixed under the action of the rotating shaft, a separating liquid is injected into the second sample processing area, the uniformly diluted sample such as the anticoagulated whole blood sample in the first sample processing area is superposed on the separating liquid and centrifuged, a target layer (such as a PBMC layer) can be seen by naked eyes after centrifugation, the target layer is sucked into the sorting sample area and added with a sheath liquid, and after the sheath liquid and the sorted sample are uniformly mixed, the chip body is driven by the rotating shaft to rotate to distribute the liquid into the sample final distribution area, so that the target sample in the sample final distribution area can be extracted, detected or collected, the complicated step of manually extracting the PBMC is omitted, the operation difficulty is reduced, the purity of the PBMC is ensured, and the target cells are prevented from being lost; the sample final distribution area is a semicircle and is arranged around the rotating shaft screens, so that the sample can be distributed to the sample final distribution area along a pipeline in the rotating process of the chip body, the distance between the second sample processing area and the rotating shaft screens is greater than the distance between the first sample processing area and the rotating shaft screens, and the sample is easier to be conveyed to the second sample processing area from the first sample processing area under the action of centrifugal force, thereby shortening the extraction time and improving the extraction efficiency of PBMCs.

Drawings

Fig. 1 is a schematic structural diagram of a butterfly microfluidic chip according to the present invention.

FIG. 2 is an enlarged schematic view of the first sample processing region, the second sample processing region, and the sorted sample region of the present invention.

Fig. 3 is an enlarged schematic view of the blocking region of the present invention.

Fig. 4 is an enlarged schematic view of a sample final distribution area according to the present invention.

FIG. 5 is a schematic representation of the stratification of an anticoagulated whole blood sample after density gradient centrifugation in the examples.

In the figure, 1, a chip body; 11. the rotating shaft is clamped; 12. a first sample treatment zone; 121. a sample mixing temporary storage cavity; 122. a sample loading hole; 123. a first ball bearing; 124. a first exhaust port; 125. a blocking region; 1251. a raised structure; 1252. a first shallow platform structure; 1253. a wax valve structure; 1254. a deep platform structure; 1255. a second shallow platform structure; 13. a second sample processing zone; 131. a first stepped laminated cavity; 132. a second vent hole; 133. a second stepped laminated cavity; 134. a third stepped laminated cavity; 135. a fourth stepped laminated cavity; 136. a fifth stepped laminated cavity; 137. a separation solution sample port; 138. a separation liquid cavity; 14. sorting the sample area; 141. a sample separation cavity; 142. a sample distribution shallow platform structure; 143. a sheath fluid injection port; 144. a second ball bearing; 145. a transition platform; 146. a deep triangular platform; 15. a sample final distribution area; 151. a distribution structure; 152. a right circular platform structure; 153. a sample collection structure; 154. a third vent hole; 16. a first thin tube; 17. a second thin tube; 18. distributing thin tubes;

2. red blood cells and sediment layer;

3. separating the liquid layer;

4. a PBMC layer;

5. serum and small molecule layers.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

In the description of the present invention, it should be understood that the terms "close to", "far away", "center", "inside (side)", "inner wall", "outside (side)", "side end", "between", "around", "input end", "output end", "left side", "right side", and the like, which indicate the orientations or positional relationships indicated in the present invention, are used based on the orientations or positional relationships shown in the drawings only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be configured and operated in a specific orientation, and thus, should not be construed as limiting the present invention.

Furthermore, it should be understood that the terms "deep" and "shallow" are used herein only for the convenience of distinguishing the structures referred to in the present invention, and are not intended to limit the actual "depth" of the structures referred to, and thus should not be construed as limiting the present invention.

The terms "first," "second," "third," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance; furthermore, unless expressly stated or limited otherwise, the terms "mounted," "connected," and "connected" are to be construed broadly, as they may be fixedly connected, detachably connected, or integrally connected, for example; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.

For the purpose of describing the working principle of the present invention, the following explanation is made: the rotating shaft matched with the rotating shaft clamping position 11 is required to realize the functions of low rotating speed, high rotating speed and left-right shaking; in addition, the position of the opening on the surface of the butterfly micro-fluidic chip corresponds to the position after the membrane is sealed, wherein the membrane sealing refers to sealing the pipeline by using single-sided pressure-sensitive adhesive, and the positions corresponding to the injection port and the exhaust hole are tightly attached to the butterfly micro-fluidic chip after being punched by a puncher; the use of a single-sided pressure sensitive adhesive has the advantages that: when the pressure-sensitive adhesive is bonded, no or little viscosity exists at an unstressed position, so that the requirement of a chip sealing pipeline processed by CNC is met; the sealing film such as pressure sensitive adhesive is adhered to the microfluidic chip, and the defects of liquid leakage and collapse are avoided when the rotating speed is increased, the ball moves and sheath liquid (and pressure) is added.

As shown in fig. 1, the present invention provides a butterfly microfluidic chip, which includes a chip body 1, wherein a rotation shaft position 11 is disposed at a center of the chip body 1, wherein the rotation shaft position 11 is adapted to an external rotation shaft and can be stably clamped without shaking, so as to prevent damage to the butterfly microfluidic chip and failure to extract PBMCs or decrease purity of extracted PBMCs, and preferably, the chip body 1 is a perfect circle to further ensure stability and balance of the chip body 1 during rotation.

Referring to fig. 1-2, in a specific example of the present invention, four large areas are disposed on a chip body 1, which are a first sample processing area 12, a second sample processing area 13, a sorting sample area 14 and a sample final distribution area 15, respectively, wherein the first sample processing area 12 is connected to the second sample processing area 13 through a first thin tube 16, the second sample processing area 13 is connected to the sorting sample area 14 through a second thin tube 17, the sorting sample area 14 is connected to the sample final distribution area 15 through a distribution thin tube 18, and a fluid medium is made to sequentially flow into the second sample processing area 13, the sorting sample area 14 and the sample final distribution area 15 through the first thin tube 16, the second thin tube 17 and the distribution thin tube 18 via the first sample processing area 12, so as to avoid manual intervention and improve extraction efficiency, and mainly: injecting a whole blood sample diluted by a proper amount into a first sample processing area 12, fully and uniformly mixing an anticoagulated whole blood sample and a diluent under the action of a rotating shaft, injecting a separating medium into a second sample processing area 13, overlapping the uniformly diluted sample in the first sample processing area 12, such as the anticoagulated whole blood sample, on the separating medium and centrifuging, wherein a target layer (such as a PBMC layer, see figure 5) can be seen by naked eyes after centrifuging, at the moment, the target layer is sucked into a sorting sample area 14 and added with a sheath liquid, and after uniformly mixing the sheath liquid and the sorted sample, the liquid is distributed into a sample final distribution area 15 along with the rotation of the rotating shaft driving a chip body 1 to rotate, so that the target sample in the sample final distribution area 15 can be extracted, detected or collected, the complicated step of manual extraction is omitted, the operation difficulty is reduced, the purity of PBMC is ensured, and the target cells are prevented from being lost; the sample final distribution area 15 is a semicircle and is arranged around the rotating shaft clamp 11, so that the sample can be distributed to the sample final distribution area 15 along a pipeline in the rotating process of the chip body 1, the distance between the second sample processing area 13 and the rotating shaft clamp 11 is greater than the distance between the first sample processing area 12 and the rotating shaft clamp 11, the speed of the sample from the first sample processing area 12 to the second sample processing area 13 is accelerated under the action of centrifugal force, the extraction time is shortened, and the extraction efficiency of PBMCs is improved.

In some examples of the present invention, as shown in fig. 2, the first sample processing area 12 includes a sample blending temporary storage cavity 121 and a first ball 123, and the first ball 123 is disposed inside the sample blending temporary storage cavity 121 and always moves along an inner wall of one end of the sample blending temporary storage cavity 121 away from a center of the chip body 1; wherein, the one end that chamber 121 is kept in to the sample mixing is close to the chip body 1 centre of a circle has seted up sample hole 122, with sample such as undiluted anticoagulation whole blood and diluent 1:1, and adding the sample into the sample mixing temporary storage cavity 121 from the position of the sample loading hole 122 after uniform mixing.

In some examples of the present invention, the size of the sample loading hole 122 is required to be sufficient for most pipette to load sample without leakage, and sample loading can be completed easily, after sample loading, the sample mixing temporary storage cavity 121 needs to be closed, and closing the sample mixing temporary storage cavity 121 means to seal the sample loading hole 122 with a sticker or other object, so as to prevent the sample from flowing backwards during rotation of the chip body 1.

Specifically, the sample for example dilutes the whole blood sample of one time and enters into sample mixing and keeps in chamber 121, and the pivot drives chip body 1 and steadily rocks from side to side, and the first ball 123 that is arranged in sample mixing and keeps in chamber 121 rocks from side to side thereupon to the mixing sample is for example dilutes the blood of one time.

In other examples of the invention, the diluent is normal saline or PBS or other diluting solution.

In an embodiment, the first ball 123 is a bead with a depth 1/3 smaller than the depth of the sample mixing temporary storage cavity 121, and is placed in the sample mixing temporary storage cavity 121 before the chip body 1 is sealed, so as to ensure that the sample mixing temporary storage cavity 121 is not adhered to the pressure sensitive adhesive, i.e. the sample mixing temporary storage cavity 121 can roll after the film is sealed, thereby achieving the effect of mixing and mixing the sample and the diluent.

In an embodiment, chamber 121 is kept in sample mixing still is equipped with first exhaust hole 124 near 1 centre of a circle one end of chip body, the sample enters into chamber 121 is kept in the sample mixing for example dilute one time's whole blood sample, the pivot drives chip body 1 and shakes about stabilizing, require the pivot rotational speed to enable the inner wall removal that the centre of a circle one end was kept away from in liquid emergence this moment, and can not flow from the position of first exhaust hole 124, first exhaust hole 124 is the inside and outside atmospheric pressure of chamber 121 is kept in order to balanced sample mixing, avoid leading to the pressure sensitive adhesive to collapse out after sealing the membrane.

In an embodiment, a blocking area 125 is disposed at an end of the sample mixing temporary storage cavity 121 away from the center of the chip body 1, and the blocking area 125 and the sample loading hole 122 are diagonally disposed on the sample mixing temporary storage cavity 121, so that the sample loading and sample unloading are located at different positions, and when the rotation shaft drives the chip body 1, the mixed sample can more easily flow out from the blocking area 125, thereby avoiding the problem that the purity of PBMCs cannot be improved due to a small amount of liquid flowing out of the mixed sample.

In one embodiment, the first venting hole 124 and the blocking area 125 are disposed on the same side, so as to ensure that the rotation speed of the rotation shaft can make the liquid move away from the inner wall of the end of the center of the circle and not flow out of the first venting hole 124, for this reason, the distance between the first venting hole 124 and the center of the circle of the chip body 1 needs to be set smaller than the distance between the blocking area 125 and the center of the circle of the chip body 1.

In a specific example of the present invention, referring to fig. 3, the blocking area 125 includes a protrusion 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, wherein the protrusion structure 1251 is disposed on an inner wall of the sample blending temporary storage cavity 121, which is far away from a center of the chip body 1, and the protrusion structure 1251 is a triangular structure, which facilitates the ball 10 to move back and forth in the sample blending temporary storage cavity 121, so as to achieve the purpose of equalizing the sample and the diluent.

In an embodiment, the first shallow platform structure 1252 is disposed in the sample blending temporary storage cavity 121 and disposed on one side of the protrusion structure 1251, so as to prevent the first ball 123 from entering the first shallow platform structure 1252 during shaking and causing blockage, and therefore the depth of the first shallow platform structure 1252 is designed to be smaller than the depth of the sample blending temporary storage cavity 121, because there is a height difference between the first shallow platform structure 1252 and the sample blending temporary storage cavity 121, the first ball 123 can be well blocked, and in order to further improve the blocking effect on the first ball 123, for this reason, the depth of the first shallow platform structure 1252 is designed to be larger than the height of the protrusion structure 1251, so that the distance between the bottom surface of the protrusion structure 1251 and the bottom surface of the first shallow platform structure 1252 is not larger than the diameter of the first ball 123, and at this time, the first ball 123 can be better limited from mistakenly entering the first shallow platform structure 1252.

With reference to fig. 3, in an embodiment, the wax valve structure 1253 is disposed outside the sample blending temporary storage cavity 121 and corresponds to the first shallow platform structure 1252, and the deep platform structure 1254 is disposed on a 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 for blocking the communication between the sample blending temporary storage cavity 121 and the second sample processing region 13, so as to prevent the sample and the diluent from entering the second sample processing region 13 when they are blended, and avoid affecting the purity of PBMC extraction.

In an embodiment, the wax valve structure 1253 does not undergo surface change at normal temperature (10 ℃ to 25 ℃), the wax valve structure 1253 begins to melt when the external temperature slowly rises to 30 ℃, the wax valve structure 1253 obviously changes when the temperature rises (does not exceed 35 ℃), and when the chip body 1 is driven by the rotating shaft to shake left and right, the uniformly mixed sample flows toward 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.

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, and when the sample, such as anticoagulated whole blood diluted by one time, enters the deep platform structure 1254, the spindle rotates counterclockwise, and since the spindle drives the chip body 1 to rotate in a single direction, the melted wax in the wax valve structure 1253 enters the deep platform structure 1254 first and fills the bottom, so that the bottom of the deep platform structure 1254 is gradually raised to a depth 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.

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 region 13, and the rotation speed ranges of 500rpm/min, 1000rpm/min, 1500rpm/min, 3000rpm/min and 5000rpm/min are tested with the increase of the centrifugal force, and the test shows that when the speed is centered, the melted wax can be spread on the bottom of the deep platform structure 1254, and can not enter the second sample processing region 13 through the first thin tube 16.

As shown in fig. 2, in some examples of the present invention, the second sample processing region 13 is disposed at the output end of the blocking region 125, that is, the liquid outlet end of the first tubule 16 is communicated with the second sample processing region 13, specifically, the second sample processing region 13 includes a plurality of step-shaped stacked cavities and a separation liquid cavity 138, the depth of the step-shaped stacked cavities increases from the end near the blocking region 125 to the end far from the blocking region 125, and the separation liquid cavity 138 is disposed at the end far from the first sample processing region 12 of the step-shaped stacked cavity.

To further explain the principle of the present invention, please refer to fig. 2 and fig. 5, a plurality of step stacked cavities are defined, and the plurality of step stacked cavities sequentially include a first step stacked cavity 131, a second step stacked cavity 133, a third step stacked cavity 134, a fourth step stacked cavity 135 and a fifth step stacked cavity 136 from a side away from a separation liquid cavity 138 to a side close to the separation liquid cavity 138, wherein the depth of the first step stacked cavity 131 is smaller than that of the second step stacked cavity 133, the depth of the third step stacked cavity 134 is smaller than that of the fourth step stacked cavity 135 is smaller than that of the fifth step stacked cavity 136, so as to form a step stacked state, which aims to enable a sample, such as a full blood sample with one-time dilution and uniform media, to diffuse away from the center of a circle and slowly and uniformly stack on the separation liquid at a low rotation speed or at a stop rotation.

In one embodiment, a second vent hole 132 is disposed on the step-stacked cavity adjacent to the first sample processing region 12 for balancing the internal and external air pressures of the second sample processing region 13, and a separation liquid loading port 137 is disposed on the separation liquid cavity 138 for loading the separation liquid into the separation liquid cavity 138.

In one embodiment, the separation liquid is a mixture of Ficoll, hydroxyethyl starch 550 and meglumine diatrizoate, or a separation liquid containing dextran (dextran) and meglumine diatrizoate as main components.

In another embodiment, the separation liquid functions on the principle of: the mononuclear cells in the peripheral blood include lymphocytes, monocytes, and the like, and have a volume, a shape, and a density different from those of other cells. The density of erythrocytes and granulocytes in blood is high, around 1.090g/ml, whereas the density of lymphocytes and monocytes is 1.075-1.090 g/ml and the density of platelets is 1.030-1.035 g/ml. After the stacking process by suitable means, the denser cells such as erythrocytes and granulocytes will settle to the bottom by the action of the centrifugation and separation liquid. In this case, leukocytes remain on the upper layer and the separation liquid layer (few number), and the material such as platelets having the lowest density is on the uppermost layer.

In some preferred embodiments, a diluted sample, such as anticoagulated whole blood, is gently stacked on the separation liquid, centrifugation is performed without mixing and shaking, a target layer, such as a PBMC layer, can be seen by naked eyes after centrifugation, the function of the stepped stacking cavity is to simulate a manual soft and soft stacking process, when the sample, such as anticoagulated whole blood diluted by one time, slowly flows from the stepped layer to the separation liquid cavity 138, and layering occurs after rotation and centrifugation of the rotating shaft; as shown in fig. 5, the serum small molecule layer 5, the PBMC layer 4, the separation liquid layer 3, and the red blood cells and sediment layer 2 are arranged in this order from top to bottom.

In some embodiments, the separation fluid is pre-loaded from the separation fluid loading port 137 and sealed with a sticker or other item that requires the separation fluid to be loaded before the wax valve is melted/before the sample, such as a double diluted anticoagulated whole blood, enters the stepped stack chamber.

In some embodiments, the depth of the first tubule 16 is the same as the depth of the second tubule 17 and the depth of the first or second shallow plateau 1252, 1255 structure.

In some embodiments, the width of the first thin tube 16 is 2 times wider than that of the second thin tube 17, because the risk that wax in the wax valve structure flows into the stepped laminated cavity after melting can be avoided, and the first thin tube 16 can be prevented from being blocked to avoid chip scrap.

In some embodiments, the lamination is necessary when the rotating shaft rotates to reach a certain rotating speed, and tests show that the rotating speed is preferably 1900rpm/min and the time duration is 5min, so that a better lamination effect can be achieved.

In some embodiments, the second thin tube 17 is disposed at the left side of the middle of the stepped stack cavity because after the sample, such as anticoagulated blood diluted twice, is centrifuged by density gradient, the PBMC layer is disposed approximately in the second stepped stack cavity 133 and the third stepped stack cavity 134, and after the sample stack is stopped, the liquid will be sucked into the sample sorting region 14 by the second thin tube 17 due to the influence of the inertia force, that is, the PBMC layer 4 and the separation liquid layer 3, etc. will flow into the sorted sample region 14 along the second thin tube 17.

In some embodiments, after the centrifugal lamination is stopped, the second air vent 132 is sealed by a sticker or adhesive tape or other articles to prevent air leakage; after the second narrow tube 17 transfers the liquid to the sorted sample area 14, the first air vent 124 in the sample mixing temporary storage cavity 121 is sealed by other articles such as a sticker or an adhesive tape, so that air is not leaked.

In other examples, the second vent hole 132 is raised upward to form an acute angle to fall down, so as to prevent backflow when filling the sorted sample region 14 with sheath fluid after standing still.

The volume of the cavity is matched with the sample loading volume and the laminated sorting volume, the volume is adjusted according to the sample loading quantity, the dilution multiple and the like when the total volume of the cavity is designed, and most of the adjustment means improvement on the depth of the cavity instead of the width.

With reference to fig. 2, in the embodiment of the present invention, the sorted sample area 14 includes a sorted sample cavity 141, a second ball 144, a transition platform 145 and a deep triangular platform 146, wherein a shallow platform structure 142 for sample distribution is disposed inside the sorted sample cavity 141, the liquid outlet end of the second thin tube 17 is communicated with the shallow platform structure 142, and the depth of the shallow platform structure 142 is shallower than that of the sorted sample cavity 141, so as to achieve the function of preventing backflow when the target layer liquid is introduced into the sorted sample cavity 141.

In some embodiments, one end of the sorted sample cavity 141 is opened with a sheath fluid injection port 143, and the sheath fluid injection port 143 needs an external associated injector to provide sheath fluid flow power; in a specific operation, a metal such as a stainless steel needle or a flat head injection head or the like slightly larger than the circumference thereof is mounted in the sheath fluid injection port 143 in advance, and is connected with the same by hot melt adhesive; the metal joint installed in the sheath fluid injection port 143 is connected to an injector containing sheath fluid such as PBS, physiological saline or other liquid medium by a hard rubber tube.

In some embodiments, the injector needs to be clamped on the injection pump to operate, so that the injector can be stably clamped, and the effect of adjusting the flow rate can be achieved.

Referring to fig. 2 and 4, in some examples of the present invention, the sample final distribution area 15 is in communication with the deep triangular platform 146 through the distribution tubule 18, the sample final distribution area 15 includes a plurality of distribution structures 151 in communication with the distribution tubule 18, the plurality of distribution structures 151 are disposed around the rotation axis position lock 11, and the sample collection structure 153 is opened with a third vent hole 154.

In some embodiments, the sheath fluid injection port 143 is always closed until the second narrow tube 17 sucks all PBMC layers in the ladder lamination cavity into the sorting sample cavity 141, and the first vent hole 124 and the second vent hole 132 in the sample mixing temporary storage cavity 121 are closed, the sheath fluid is slowly added, and the third vent hole 154 is always open when the sheath fluid is injected.

In some embodiments, the second ball 144 is disposed inside the sorted sample cavity 141, and is used for uniformly mixing the sheath fluid and the sorted sample, and as the rotating shaft drives the chip body 1 to rotate to distribute the fluid to the transition platform 145, the second ball 144 and the first ball 123 are used in the same manner, and the rotating shaft is required to drive the chip body to rotate left and right; the difference from the first ball 123 is that the second ball 144 is used for uniformly mixing the sheath fluid and the sorted sample without an absolute uniform medium, and only has a slight uniform mixing effect.

In some embodiments, the transition platform 145 is disposed at an end of the sorted sample cavity 141 away from the sheath fluid inlet 143, and the deep triangular platform 146 is disposed at a side of the transition platform 145 away from the sorted sample cavity 141, wherein a depth of the transition platform 145 is identical to a depth of the sorted sample cavity 141, and serves as a buffer for the sheath fluid and the sorted sample, and a depth of the deep triangular platform 146 is deeper than a depth of the transition platform 145, and serves to connect with the deeper distribution tubules 18, so as to distribute the sample into the respective distribution structures 151.

In some embodiments, the sheath fluid is used for providing external power for the sample mixed solution obtained by sorting, and the liquid obtained in the sample sorting cavity is subjected to next distribution; the sample drives sheath liquid to flow in from the sheath liquid sample inlet 143 due to the pressure of the external injection pump, and then the sample is transferred to the deep triangular platform 146 due to the flowing direction of the sheath liquid and the action of centrifugal force; after that, the chip does not rotate along with the rotating shaft any more and is in a static state.

In other examples, the chip may be placed on a horizontal surface and subsequent power may be derived from the sheath fluid only after the syringe containing the sheath fluid is connected.

The volume of the cavity is three times or more than the original sample volume, the function of the cavity is to meet the requirement that sufficient space can be realized during sheath liquid sample introduction, and the sealing membrane is not broken when the sheath liquid sample introduction speed is higher.

The sample and the sheath fluid mixed solution that obtain of sorting along with the inflow of sheath fluid introduction port sheath fluid, distribute to each distribution structure 151 along distributing tubule 18 in proper order, wherein, distributing tubule 18 sets to semi-circular structure, make liquid for example after the mixed solution of sample and sheath fluid that obtains of sorting fills up first distribution structure 151 earlier, just can enter into second distribution structure 151 and fill, so on, avoid leading to subsequent drawing, detection or collection to be influenced because of distributing inhomogeneously, thereby improve PBMC's purity.

In other examples, the end of the distribution tubule 18 is provided with a circular platform structure 152, the circular platform structure 152 is circumferentially provided with a sample collection structure 153, sheath fluid is continuously added along with the extension of time, the volume of the liquid rises after the sorted sample is continuously diluted, and the liquid enters the circular platform structure 152 and the sample collection structure 153, wherein the circular platform structure 152 can play a role in preventing backflow, and the sample collection structure 153 can be temporarily stored as waste liquid or can be used as a final reaction area.

In other examples, the sum of the total volume of the cavities and the total volume of the circular sample collection structure is required not to exceed the maximum volume, so as to avoid the collapse of the sealing membrane or the liquid flowing out of the third vent hole 154 due to the addition of the sheath liquid;

in other examples, the depth of the third vent 154 should be set shallow to avoid affecting the normal function of the sample collection structure 153.

In other examples, a metal needle is inserted into the sheath fluid inlet 143, which is also positioned symmetrically around the center of the circle to balance the centrifugal forces at the two ends.

The working process of the invention is as follows:

1. a sample such as a double-diluted whole blood sample enters the sample mixing temporary storage cavity 121, the rotating shaft drives the chip body 1 to shake, and the first ball 123 in the sample mixing temporary storage cavity 121 shakes therewith, so that the sample such as the double-diluted whole blood sample is mixed, the first vent hole 124 is opened after the sample is added, and the sample loading hole 122 is sealed;

2. the melted wax in the wax valve structure 1253 firstly enters the deep platform structure 1254 and is filled below, the sample flows into the second shallow platform structure 1255 after passing through the first shallow platform structure 1252 and the deep platform structure 1254 and then enters the first stepped laminated cavity 131 through the first thin tube 16, before the wax valve is melted at a temperature/before the sample enters the stepped laminated cavity, for example, anticoagulated whole blood diluted by one time is added with a separation solution from the separation solution sample inlet 137, the separation solution sample inlet 137 is sealed, and the second vent hole 132 is opened;

3. a sample such as anticoagulated whole blood diluted by one time enters the stepped laminated cavity for centrifugal layering, and after the centrifugal layering is static, the second exhaust hole 132 is sealed and does not leak air; after the second thin tube 17 transfers the target layer such as the PBMC layer to the sorting sample area 14 through the second thin tube 17, the first vent hole 124 is sealed;

4. the PBMC layers in the stepped laminated cavity are all sucked into the sorting sample cavity 141, after being uniformly mixed through the second ball 144, and under the condition that the first exhaust hole 124 and the second exhaust hole 132 are closed, sheath liquid is slowly added, the third exhaust hole 154 is always in an open state, a sample drives the sheath liquid to flow from the sheath liquid inlet 143 to the transition platform 145 due to the pressure of an external injection pump, and then the sample is transferred to the deep triangular platform 146 due to the flowing direction of the sheath liquid and the action of centrifugal force;

5. the mixed solution of the sorted sample and the sheath fluid is sequentially distributed to each distribution structure 151 along the distribution tubule 18 along with the inflow of the sheath fluid from the sheath fluid injection port, wherein the distribution tubule 18 is set to be a semicircular structure, so that the liquid, such as the mixed solution of the sorted sample and the sheath fluid, can enter the second distribution structure 151 for filling after the first distribution structure 151 is filled with the mixed solution, and finally reaches the perfect circular platform structure 152 and the sample collection structure 153.

In summary, according to the butterfly-type microfluidic chip provided by the invention, after the whole blood sample is injected into the first sample processing region 12, the target sample can be extracted, detected or collected in the sample final distribution region 15, so that the tedious step of manually extracting PBMCs is omitted, the operation difficulty is reduced, the purity of PBMCs is ensured, the target cells are prevented from being lost, the extraction time can be shortened, and the extraction efficiency of PBMCs is improved.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and substitutions can be made without departing from the technical principle of the present invention, and these modifications and substitutions should also be regarded as the protection scope of the present invention. Having shown and described the basic principles, essential features and advantages of the invention, it will be apparent to those skilled in the art that it is not restricted to the details of the preferred embodiments described above, which are to be regarded as illustrative rather than restrictive, the scope of the invention being defined by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Furthermore, it should be understood that although the specification describes embodiments, not every embodiment includes only a single embodiment, and such description is for clarity purposes only, and it will be understood by those skilled in the art that the specification as a whole and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims (10)

1. A butterfly type micro-fluidic chip is characterized by comprising a chip body, wherein a rotating shaft clamping position is arranged at the center of the chip body, and a first sample processing area, a second sample processing area, a sorting sample area and a sample final distribution area are respectively arranged on the chip body; wherein the first sample processing region is piped to the second sample processing region, the second sample processing region is piped to the sort sample region, the sort sample region is piped to the sample final distribution region, the sample final distribution region is semi-circular and is arranged around the spindle detent, and the distance between the second sample processing region and the spindle detent is greater than the distance between the first sample processing region and the spindle detent.

2. The butterfly microfluidic chip of claim 1, wherein the first sample processing region comprises:

a sample mixing temporary storage cavity, wherein a sample loading hole is formed in one end, close to the circle center of the chip body, of the sample mixing temporary storage cavity, a blocking area is formed in one end, far away from the circle center of the chip body, of the sample mixing temporary storage cavity, and the blocking area and the sample loading hole are arranged on the sample mixing temporary storage cavity in a diagonal manner; and

the first ball is arranged in the sample mixing temporary storage cavity and always follows the sample mixing temporary storage cavity to be kept away from the inner wall of one end of the circle center of the chip body.

3. The butterfly-type microfluidic chip according to claim 2, wherein a first vent hole is further formed in the sample mixing temporary storage cavity, the first vent hole and the blocking area are arranged on the same side, and a distance between the first vent hole and a circle center of the chip body is smaller than a distance between the blocking area and the circle center of the chip body.

4. The butterfly microfluidic chip of claim 2, wherein the diameter of the first ball is less than one third of the depth of the sample mixing buffer chamber.

5. The butterfly microfluidic chip of claim 2, wherein the blocking region comprises:

the protruding structure is arranged on the inner wall of the sample blending temporary storage cavity, which is far away from the inner wall of the circle center of the chip body;

the first shallow platform structure is arranged in the sample blending temporary storage cavity and is arranged on one side of the bulge structure;

the wax valve structure is arranged on the outer side of the sample blending temporary storage cavity and corresponds to the first shallow platform structure;

the deep platform structure is arranged on one side, away from the first shallow platform structure, of the wax valve structure; and

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

6. The butterfly microfluidic chip of claim 5, wherein the depth of the first shallow platform structure is smaller than the depth of the sample mixing temporary storage cavity, the depth of the first shallow platform structure is smaller than the height of the protrusion structure, and the distance between the bottom surface of the protrusion structure and the bottom surface of the first shallow platform structure is not larger than the diameter of the first ball.

7. The butterfly microfluidic chip according to claim 2, wherein the second sample processing region is disposed at an output end of the blocking region, the second sample processing region includes a plurality of step stack cavities and a separation liquid cavity, a depth of each step stack cavity increases gradually from a position close to the blocking region to a position away from the blocking region, and the separation liquid cavity is disposed at an end of each step stack cavity away from the first sample processing region.

8. The butterfly microfluidic chip of claim 7, wherein a second vent hole is disposed on the stepped stacked cavity adjacent to the first sample processing region, and a separation solution sample port is disposed on the separation solution cavity.

9. The butterfly microfluidic chip of claim 7, wherein the sorted sample region comprises:

the separation device comprises a separation sample cavity, wherein a sample distribution shallow platform structure is arranged on the inner side of the separation sample cavity and communicated with a stepped laminated cavity close to one side of the separation liquid cavity through a second thin tube, and one end of the separation sample cavity is provided with a sheath liquid injection port;

the second ball is arranged on the inner side of the sorting sample cavity;

the transition platform is arranged at one end, far away from the sheath fluid injection port, of the sorted sample cavity; and

the deep triangular platform is arranged on one side, far away from the sorting sample cavity, of the transition platform.

10. The butterfly type microfluidic chip according to claim 9, wherein the final sample distribution area is communicated with the deep triangular platform through a distribution tubule, the final sample distribution area includes a plurality of distribution structures communicated with the distribution tubule, the plurality of distribution structures are disposed around the rotation axis, a circular platform structure is disposed at an end of the distribution tubule, a sample collection structure is disposed in a circumferential direction of the circular platform structure, and a third vent hole is disposed in the sample collection structure.

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