Erythrocyte separation structure, biochip, device and method

Abstract

The invention discloses a red blood cell separation structure, a biological chip, a device and a method.The red blood cell separation structure comprises a first cavity, the first cavity is provided with a first sample adding opening, a slope is arranged at the bottom of the first cavity, and a solid reagent is contained in the first cavity; the solid reagent comprises a magnetic bead and an antibody which is adsorbed on the surface of the magnetic bead and is used for adsorbing red blood cells, and a permanent magnet can be adsorbed on the side wall of the first chamber. According to the chamber structure design for separating the red blood cells, complete separation of plasma and the red blood cells is achieved. The purpose of the invention is to solve the problems of whole blood filtration of a large sample size and hemolysis of a high-pressure-volume sample.

Description

Technical Field

[0001] The present invention relates to the field of biological instruments, and particularly to a red blood cell separation structure, a biochip, a device and a method. Background Art

[0002] In the in vitro diagnosis (IVD) industry, most of the samples used for detection are plasma / serum. The samples collected by hospital medical staff from patients are whole blood, which cannot be directly used for detection because red blood cells, white blood cells, and other fibrous impurities in it will seriously affect the accuracy of the detection results. The traditional method for processing whole blood is the centrifugation method. The disadvantages of centrifugation are that it requires additional equipment and a long processing time. Currently, some rely on membrane filtration. The simple membrane filtration method has high requirements for the membrane material and process, is difficult to produce, and only imported membranes can be used, resulting in high costs. Moreover, the membrane will adsorb a certain amount of plasma, thereby reducing the sample utilization rate.

[0003] Chinese Utility Model Patent CN210079266U discloses a structure for lateral separation of red blood cells. The structure for lateral separation of red blood cells includes a substrate, a first assembly cavity and a second assembly cavity arranged in the substrate, and a sample addition port and a collection cavity extending downward from the top surface of the substrate; the first assembly cavity is used to place a first filter element, the second assembly cavity is used to place a second filter element, the sample addition port, the first assembly cavity, the second assembly cavity and the collection cavity are connected in series; the second filter element is vertically arranged to form a lateral filter element. However, when using the filter membrane filtration method for specimens with a large hematocrit, it is easy to cause the whole blood sample to block the blood filter membrane, block the liquid driving force, prevent the plasma from flowing in a preset manner, and even cause red blood cell rupture and hemolysis, affecting the test results; the blood filter membrane needs to be strictly sealed during use to prevent red blood cells from leaking through the side, and the bonding process is complex. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a red blood cell separation structure, a biochip, and a device to solve the problem that the filter membrane filtration method is prone to block the filter membrane with whole blood samples when the hematocrit is large, resulting in red blood cell rupture and hemolysis, aiming at the deficiencies of the prior art.

[0005] The present invention also provides a method for separating red blood cells to solve the problem that the filter membrane filtration method is prone to block the filter membrane with whole blood samples when the hematocrit is large.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is: a red blood cell separation structure, including a first chamber, the first chamber is provided with a first sample addition port, the bottom of the first chamber is provided with a slope, and the first chamber is filled with a solid reagent, and the solid reagent includes magnetic beads and an antibody adsorbed on the surface of the magnetic beads for adsorbing red blood cells.

[0007] The chamber structure design for separating red blood cells in the present invention realizes the complete separation of plasma and red blood cells. The purpose of this solves the problems of whole blood filtration with a large sample size and hemolysis of samples with a high hematocrit.

[0008] The solid reagent in the present invention is made by using the immunomagnetic bead technology. The immunomagnetic bead technology (Immunomagnetic bead, IMB) performs antigen-antibody reactions by conjugating and coating antibodies on the surface of magnetic beads, binds to the surface of antigens to form immune complexes, and separates the antigen-antibody-magnetic bead immune complexes under the action of strong magnetism to achieve antigen screening or removal of labeled antigens. By using the immunomagnetic bead technology to rapidly separate red blood cells to obtain plasma and applying it to immunological detection, it can better serve clinical diagnosis. For example, in the existing patent technology, Chinese Patent Application No. CN200710305363.6 discloses a method for separating red blood cells in whole blood using magnetic microspheres coated with lectin or anti-red blood cell antibodies.

[0009] In a preferred embodiment of the present invention, in order to facilitate the aspiration of plasma after separation and ensure that only plasma is aspirated when adding samples and aspirating plasma, a guiding structure is provided at the first sample addition port, and the lower end of the guiding structure is far from one of the side walls of the first chamber.

[0010] In a preferred embodiment of the present invention, in order to aspirate plasma to the greatest extent and reduce plasma residue, the guiding structure is a frustum-shaped pipe.

[0011] In a preferred embodiment of the present invention, in order to ensure that only plasma is aspirated when adding samples and aspirating plasma, the guiding structure is close to the lower end of the slope.

[0012] In a preferred embodiment of the present invention, in order to facilitate the separation of plasma and red blood cells, the angle of the slope is 10-20°.

[0013] In a preferred embodiment of the present invention, in order to reduce liquid residue, rounded corners are provided at the periphery of the slope.

[0014] In a preferred embodiment of the present invention, the red blood cell separation structure further includes a filtering structure.

[0015] In a preferred embodiment of the present invention, the filtering structure includes a second chamber, a plunger is plugged at the upper end of the second chamber, the side wall of the second chamber is communicated with a second sample addition structure, an outlet pipeline is provided at the outlet of the second chamber, and a filter membrane is provided at the liquid inlet end of the outlet pipeline.

[0016] In this process, substances such as white blood cells and plasma fibers that affect the measured value in the plasma will be intercepted. The filter membrane only needs to filter white blood cells, reducing the requirements for the membrane.

[0017] Since the red blood cell separation and filtration are independently separated, the time for each step is shortened, and they can be carried out step by step synchronously, making the process faster.

[0018] In a preferred embodiment of the present invention, a quantitative nozzle is further provided at the liquid outlet end of the outlet pipeline; the second sample adding structure is inclined.

[0019] The present invention also discloses a biochip, including the red blood cell separation structure described above.

[0020] The present invention also discloses a device for separating red blood cells, including the biochip and a permanent magnet for adsorbing magnetic beads, and the permanent magnet is close to or adjacent to the side wall of the first chamber.

[0021] In a preferred embodiment of the present invention, the lower end of the side wall of the first chamber is away from the guiding structure;

[0022] The device for separating red blood cells further includes a driving mechanism for driving the plunger.

[0023] The lower end of the side wall away from the guiding structure ensures that when adding samples and aspirating plasma, it deviates from the side of the permanent magnet.

[0024] The permanent magnet corresponds to the position of the first chamber, and a driving structure can be set to control the permanent magnet to be close to or away from the side wall of the first chamber.

[0025] The present invention also discloses a method for separating red blood cells, using the above device to separate red blood cells, including the following steps:

[0026] S1. Add the whole blood sample into the first chamber, mix well and let it stand;

[0027] S2. Adsorb the permanent magnet on the side wall of the first chamber;

[0028] S3. Aspirate the plasma at the lower end of the slope.

[0029] In a preferred embodiment of the present invention, the method for separating red blood cells further includes the following steps:

[0030] S4. Add the plasma aspirated in S3 into the second chamber through the second sample adding structure, drive the plunger to press down, so that the plasma passes through the filter membrane and then is added into the third chamber to complete the filtration of the whole blood sample.

[0031] In a preferred embodiment of the present invention, the standing time in S1 is 30 - 60S;

[0032] The time for the permanent magnet to be adsorbed on the side wall of the first chamber in S2 is 30 - 150S.

[0033] After standing still for 30 to 60 seconds, the magnetic particles in the solid reagent will specifically bind to the red blood cells in the whole blood.

[0034] After magnetic absorption for 30 to 150 seconds, all the plasma and red blood cells will be separated.

[0035] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0036] 1. Fast speed, reducing the requirements for the filtration membrane, including the requirements for the membrane material and area;

[0037] 2. Reducing the requirements for the membrane, including the requirements for the membrane material and area;

[0038] 3. Avoiding the hemolysis of the whole blood under high pressure;

[0039] 4. Facilitating the realization of automation;

[0040] 5. The biochip can achieve the filtration of the whole blood with a large sample size;

[0041] 6. After the red blood cell separation chamber and the plasma filtration chamber are separated independently, the functional requirements realized by each chamber are simplified, and the requirements for the instrument are lower. In the prior art, the red blood cells are also separated in the filtration chamber, so the instrument needs to monitor the hemolysis, and the intelligent requirements for filtration are higher. But in this current scheme, the above-mentioned hardware and control requirements are not needed, making it easier for the instrument to achieve automation. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is a schematic structural diagram of the red blood cell separation structure in an embodiment of the present invention after removing the cover glass A and the plunger.

[0043] Figure 2 It is a schematic structural diagram of the red blood cell separation structure in an embodiment of the present invention after removing the cover glass B.

[0044] Figure 3 It is a schematic structural assembly diagram of the red blood cell separation structure in an embodiment of the present invention.

[0045] Figure 4 It is a schematic structural assembly diagram of the device for separating red blood cells in an embodiment of the present invention.

[0046] Figure 5 It is a three-dimensional structural schematic diagram of the red blood cell separation structure in an embodiment of the present invention after removing the cover glass A.

[0047] Among them, 1. Cover sheet A; 2. Substrate; 3. Cover sheet B; 4. Second sample adding structure; 5. Second chamber; 6. First chamber; 7. Permanent magnet; 8. Guide structure; 9. Solid reagent; 10. Plunger; 11. Filter membrane; 12. Outlet pipeline; 13. Quantitative nozzle; 14. First sample adding port; 15. Ramp. Detailed implementation manner

[0048] Such as Figures 1 - 3 , Figure 5 shown, a red blood cell separation structure is composed of three parts: cover sheet A1, substrate 2, and cover sheet B3. The first chamber 6 can be a sealed chamber composed of the substrate 2 and cover sheet B3. The first chamber 6 can also be designed as a sealed chamber composed of the substrate 2, cover sheet A1, and cover sheet B3. The first chamber 6 is provided with a first sample adding port 14, and the bottom of the first chamber 6 is provided with a ramp 15, and the angle of the ramp 15 is 10 - 20°. A guide structure 8 is provided at the first sample adding port 14, and the lower end of the guide structure 8 is far from the side wall of the first chamber 6 that adsorbs the permanent magnet 7, so that when adding samples and aspirating plasma, it deviates from the side of the permanent magnet 7. The guide structure 8 is a frustum-shaped pipe. The guide structure 8 is close to the lower end of the ramp 15. Fillets are provided around the ramp 15 to reduce liquid residue. The first chamber 6 is filled with a solid reagent 9, and the solid reagent includes magnetic beads and antibodies adsorbed on the surface of the magnetic beads for adsorbing red blood cells. The side wall of the first chamber 6 can adsorb the permanent magnet 7.

[0049] The red blood cell separation structure further includes a filtering structure. The filtering structure includes a second chamber 5, the upper end of the second chamber 5 is plugged with a plunger 10, the side wall of the second chamber 5 is communicated with a second sample adding structure 4, the second sample adding structure 4 is inclined, and the second sample adding structure 4 is penetrated through the ramp to the second chamber 5. An outlet pipeline 12 is provided at the outlet of the second chamber 5, and a filter membrane 11 is provided at the liquid inlet end of the outlet pipeline 12. A quantitative nozzle 13 is further provided at the liquid outlet end of the outlet pipeline 12. Among them, the outlet of the second chamber 5 and the outlet pipeline 12 form a closed pipeline composed of the substrate 2 and cover sheet A1. The permanent magnet 7 is a component on the instrument and is a movable component. It will be closely attached to the side of the cover sheet B3 when needed.

[0050] A device for separating red blood cells includes the biochip and a permanent magnet 7 for adsorbing magnetic beads, and the permanent magnet 7 is adsorbed on the side wall of the first chamber 6. The device for separating red blood cells further includes a driving mechanism for driving the plunger 10.

[0051] The working process of this solution:

[0052] When starting to process whole blood, the permanent magnet 7 is not close to the chip. The whole blood can be added to the first chamber 6 from the guiding structure 8 manually or by an instrument. The solid reagent 9 will dissolve when it encounters the whole blood. After adding the whole blood, the solid reagent and the whole blood are fully reacted by mixing. The mixing methods include pipetting and mixing, ultrasonic mixing, and mechanical mixing. In this example, pipetting and mixing is preferably used. After mixing, let it stand for reaction for 30 - 60S. During the standing process, the magnetic particles in the solid reagent will specifically bind to the red blood cells in the whole blood.

[0053] After the standing is completed, the instrument will move the permanent magnet 7 from a position far from the chip to close to the chip cover glass B3 (as Figure 4 shown), and then all the red blood cells in the first chamber 6 will be adsorbed onto the cover glass B3 along with the magnetic particles in the solid reagent 9. The plasma and red blood cells in the whole blood will be separated. Because of the different hematocrits of the red blood cells, after 30 - 150S of magnetization, all the plasma and red blood cells will be separated. After separation, the plasma is aspirated into the pipette tip manually or by an instrument. Because of the guiding structure 8 of the first chamber 6, it can ensure that the pipette tip is on the side far from the permanent magnet 7 at the bottom, and what is aspirated is all plasma. Coupled with the ramp structure of the first chamber 6, the plasma will automatically gather around the pipette tip, enabling the maximum aspiration of plasma and reducing plasma residue.

[0054] After extracting the plasma, move the pipette tip above the second sample addition structure 4 and add it to the second chamber 5 through the second sample addition structure 4. After adding the plasma, the instrument presses down the plunger 10. As the plunger 10 is pressed down, the plasma is quantitatively dispensed under pressure through the filter membrane 11, the outlet pipeline 12, and the metering nozzle 13. Substances such as white blood cells and plasma fibrin that affect the measured value in the plasma will be intercepted during this process.

Claims

1. A red blood cell separation structure, characterized in that: It includes a first chamber (6), the first chamber (6) is provided with a first sample adding port (14), the bottom of the first chamber (6) is provided with a slope (15), the first chamber (6) is filled with a solid reagent (9), and the solid reagent includes magnetic beads and antibodies adsorbed on the surface of the magnetic beads for adsorbing red blood cells.

2. The red blood cell separation structure according to claim 1, characterized in that: A guiding structure (8) is arranged at the first sample adding port (14), and the lower end of the guiding structure (8) is far away from one side wall of the first chamber (6).

3. The red blood cell separation structure according to claim 2, wherein: The guiding structure (8) is a frustum-shaped pipe.

4. The erythrocyte separation structure according to claim 2, wherein: The guiding structure (8) is close to the lower end of the slope (15).

5. The red blood cell separation structure according to claim 1, wherein: The angle of the slope (15) is 10-20°.

6. The red blood cell separation structure according to claim 1, wherein: Round corners are provided at the periphery of the slope (15).

7. The red blood cell separation structure according to any one of claims 1-6, characterized in that: It further includes a filtering structure.

8. The erythrocyte separation structure according to claim 7, wherein: The filtering structure includes a second chamber (5), a plunger (10) is plugged at the upper end of the second chamber (5), the side wall of the second chamber (5) is communicated with a second sample adding structure (4), an outlet pipeline (12) is arranged at the outlet of the second chamber (5), and a filter membrane (11) is arranged at the liquid inlet end of the outlet pipeline (12).

9. The red blood cell separation structure according to claim 8, wherein: A quantitative nozzle (13) is further arranged at the liquid outlet end of the outlet pipeline (12); The second sample adding structure (4) is arranged obliquely.

10. A biochip, characterized in that, It includes the red blood cell separation structure according to any one of claims 1-9.

11. An apparatus for separating red blood cells, characterized in that, It includes the biochip according to claim 10 and a permanent magnet (7) for adsorbing magnetic beads, and the permanent magnet (7) is close to or adjacent to the side wall of the first chamber (6).

12. The apparatus for separating red blood cells according to claim 11, characterized in that, The side wall of the first chamber (6) is far away from the lower end of the guiding structure (8); The device for separating red blood cells further includes a driving mechanism for driving the plunger (10).

13. A method for separating red blood cells, characterized in that, Using the device according to claim 11 to separate red blood cells, includes the following steps: S1. Add a whole blood sample into the first chamber (6), mix well and let it stand; S2. Adsorb the permanent magnet (7) on the side wall of the first chamber (6); S3. Aspirate the plasma at the lower end of the slope (15).

14. The method for separating red blood cells according to claim 13, wherein, It further includes the following steps: S4. Add the plasma aspirated in S3 to the second chamber (5) through the second sample adding structure (4), drive the plunger (10) to press down, so that the plasma passes through the filter membrane (11) and then is added to the third chamber (10) to complete the filtration of the whole blood sample.

15. The method for separating red blood cells according to claim 13, characterized in that, The standing time in S1 is 30-60S; The adsorption time of the permanent magnet (7) on the side wall of the first chamber (6) in S2 is 30-150S.

Images