Microfluidic cell separation and detection device

Abstract

The utility model provides a microfluidic cell separation and detection device, and belongs to the field of cell detection. The device comprises a case, wherein a separation unit, a heating unit, a control system and a fluorescence detection module are arranged on the case; the heating unit and the fluorescence detection module are connected with the separation unit and the control system; the separation unit comprises a chip fixing frame and a micro-fluidic chip embedded in the chip fixing frame; the micro-fluidic chip is provided with a liquid inlet channel, an annular combination pool and a liquid outlet channel, the liquid inlet channel is provided with a liquid inlet, the liquid inlet channel is correspondingly communicated with the liquid inlet side of the annular combination pool, the liquid outlet channel is provided with a waste liquid discharge hole, and the liquid outlet channel is correspondingly communicated with the liquid outlet side of the annular combination pool; the heating module is arranged between the chip fixing frame and the annular combination pool; the annular combination pool is provided with a plurality of separation reaction cavities and mineral oil adding holes which are communicated with one another. According to the utility model, various cell types can be efficiently and specifically separated and detected, meanwhile, the operation process is simplified, and the analysis efficiency and accuracy are improved.

Description

Technical Field

[0001] The utility model relates to the field of cell detection, in particular to a microfluidic cell separation detection device. Background Art

[0002] Cell separation technology plays a key role in biomedical research, clinical diagnosis and treatment. With the rapid development of precision medicine and single-cell analysis, separation technologies that are efficient, specific and maintain cell integrity are becoming increasingly important. Traditional cell separation methods mainly include density gradient centrifugation, magnetic activated cell sorting (MACS), fluorescence activated cell sorting (FACS) and adhesion separation. Density gradient centrifugation uses cell density differences for separation, which is simple to operate but has limited resolution; MACS uses magnetic beads to label specific cell surface antigens and achieves separation in a magnetic field, which has high specificity but may have non-specific binding; FACS is a high-throughput, multi-parameter cell sorting technology that can analyze multiple cell characteristics at the same time, but the equipment is expensive and the operation is complicated; adhesion separation uses the difference in the adhesion ability of different cells to the culture surface for separation, which is simple but has low specificity. In addition, there are some emerging cell separation technologies, such as acoustic separation and optical tweezers separation. These methods have their own advantages and disadvantages in practical applications, and researchers have been seeking more efficient, specific and less damaging separation technologies for cells. At present, the main challenges facing cell separation technology include: how to maintain high separation efficiency while maximally maintaining cell activity and functional integrity; how to achieve simultaneous separation of multiple cell types; how to seamlessly integrate cell separation with subsequent analysis (such as PCR, sequencing, etc.). In addition, cell separation technology also needs to consider factors such as automation of sample processing, reproducibility of results, and ease of operation.

[0003] Chinese invention patent CN202121465016.1 proposes a multiple immunoaffinity cell capture device based on a microfluidic chip. The device consists of three independent microfluidic chambers, each of which is modified with different specific antibodies on the inner surface to capture different types of target cells. This patent has the following disadvantages:

[0004] 1. Because it is a negative selection method, only one type of cell can be screened out;

[0005] 2. Lack of circulation flow mechanism may affect the full contact between cells and antibodies;

[0006] 3. There is no ability to integrate subsequent molecular analysis (such as PCR), and the captured cells need to be transferred to other devices for analysis, which increases operational complexity and the risk of sample loss;

[0007] 4. It does not have in situ PCR and detection functions, and requires additional equipment and steps, which increases analysis time and cost;

[0008] 5. It is difficult to achieve effective cyclic cleaning of captured cells, which may affect the accuracy of subsequent analysis;

[0009] 6. Failure to consider how to prevent cross contamination between different reaction areas;

[0010] 7. The cavity design limits the ability to further expand to capture more types of cells.

[0011] Prior art "Study of flow behaviors on single-cell manipulation and shear stress reduction in microfluidic chips using computational fluiddynamics simulations" proposed an integrated microfluidic device for the capture of circulating tumor cells (CTCs) and subsequent gene expression analysis. The device combines microfluidic immunoaffinity capture technology and in situ RNA analysis functions, but the device has the following disadvantages:

[0012] 1. Only a single type of cell (CTCs) is captured, making it difficult to separate multiple cell types at the same time;

[0013] 2. Lack of effective cleaning steps may affect the accuracy of subsequent gene expression analysis;

[0014] 3. The bubble-driven microvalve system may have stability issues, affecting the accuracy of liquid control;

[0015] 4. The chip design is relatively fixed and difficult to flexibly adjust according to different experimental requirements;

[0016] 5. Failure to consider how to deal with nonspecific binding issues, which may affect the specificity of the analysis;

[0017] 6. Lack of circulation flow mechanism may affect the full contact between cells and antibodies;

[0018] 7. Direct cell lysis in the capture area may lead to the release of non-target RNA, affecting the specificity of subsequent analysis.

[0019] In recent years, the development of microfluidics has provided new ideas and methods for cell separation. Microfluidics uses the fluid mechanics of micron-scale channels to achieve precise manipulation and separation of cells. Cell separation methods based on microfluidics mainly include inertial microfluidics separation, acoustic separation, dielectrophoresis separation, and microfluidic immunoaffinity separation. Among them, inertial microfluidics separation uses the balance of inertial force and secondary flow (also known as Dean secondary flow) in microchannels to achieve separation according to cell size; acoustic separation uses acoustic force fields to manipulate cells; dielectrophoresis separation uses the polarization effect of cells in non-uniform electric fields for separation; microfluidic immunoaffinity separation modifies antibodies in microchannels to specifically capture target cells. These cell separation methods based on microfluidics have the advantages of simple operation, low sample consumption, and high separation efficiency, and show greater advantages than traditional methods in many application scenarios. In this context, the present application provides a new cell separation device that can overcome the limitations of the prior art. Utility Model Content

[0020] In response to the problems existing in the above-mentioned prior art, the utility model provides a microfluidic cell separation and detection device, which aims to improve the simultaneous separation efficiency of multiple cell types and realize the efficient capture of different target cells in complex biological samples; enhance the integration of cell separation and subsequent molecular analysis, reduce sample transfer and processing steps, and reduce sample loss and contamination risks; improve the specificity and sensitivity of cell capture, especially for the enrichment and detection of low-abundance cells; simplify the operational procedures of cell separation and molecular detection, improve the degree of automation of the experiment and the repeatability of the results; realize integrated operation from cell separation to nucleic acid detection, and improve analysis efficiency and accuracy; optimize the use efficiency of samples and reagents, reduce experimental costs and resource consumption; enhance the adaptability and flexibility of the equipment so that it can be suitable for different types of biomedical research and clinical diagnosis needs.

[0021] In order to solve the above technical problems, the utility model provides a microfluidic cell separation and detection device, comprising a chassis, on which a separation unit, a heating unit, a control system and a fluorescence detection module are arranged; the heating unit and the fluorescence detection module are both electrically connected to the separation unit and the control system;

[0022] The separation unit comprises a microfluidic chip and a chip fixing frame, wherein the microfluidic chip is embedded in the chip fixing frame; the microfluidic chip is provided with a liquid inlet channel, a binding pool and a liquid outlet channel, wherein the binding pool has a liquid inlet side and a liquid outlet side, wherein the liquid inlet channel has a liquid inlet port, wherein the liquid inlet channel is correspondingly connected to the liquid inlet side of the binding pool, wherein the liquid outlet channel has a waste liquid discharge hole, wherein the liquid outlet channel is correspondingly connected to the liquid outlet side of the binding pool;

[0023] The heating unit comprises a heating module, and the heating module is arranged between the chip fixing frame and the binding pool;

[0024] The combination pool is provided with a plurality of separation reaction chambers and mineral oil adding holes which are interconnected.

[0025] Furthermore, the liquid inlet channel includes a sample liquid inlet channel, a cleaning liquid inlet channel and a PCR reaction liquid inlet channel; the sample liquid inlet channel has a sample addition hole, the cleaning liquid inlet channel has a cleaning liquid addition hole, and the PCR reaction liquid inlet channel has a PCR reaction liquid addition hole; the sample liquid inlet channel, the cleaning liquid inlet channel and the PCR reaction liquid inlet channel are connected to the corresponding liquid inlet side of the binding pool after merging.

[0026] Furthermore, the sample addition hole, the cleaning solution addition hole, the PCR reaction solution addition hole and the waste liquid discharge hole are all provided with valves accordingly.

[0027] Furthermore, the binding pool is annular.

[0028] Furthermore, the combination pool has an annular channel, one side of the annular channel is connected to the liquid inlet channel, and the other side of the annular channel is connected to the liquid outlet channel.

[0029] Furthermore, a piezoelectric micro pump is connected to the annular channel, and the piezoelectric micro pump is electrically connected to the control system.

[0030] Furthermore, a plurality of separation reaction chambers are arranged on the annular channel, a mineral oil adding hole is arranged on the inner side of the annular channel, and the mineral oil adding hole is connected to a plurality of delivery channels communicating with the annular channel.

[0031] Furthermore, four separation reaction chambers are evenly arranged on the annular channel.

[0032] Furthermore, the mineral oil adding hole is connected to four delivery channels which are in communication with the annular channel.

[0033] Furthermore, the heating unit also includes a temperature control system, which is connected to the heating module, disposed in the chassis, and electrically connected to the control system.

[0034] Furthermore, the heating module is in the shape of a honeycomb point.

[0035] Furthermore, the chassis is also provided with a lock that cooperates with the fluorescence detection module.

[0036] Furthermore, the chip fixing frame is provided with a recess, and the microfluidic chip is fixed in the recess.

[0037] The utility model provides a microfluidic cell separation detection device, which has at least the following beneficial effects:

[0038] 1. By delivering pre-mixed different labeled antibodies to the annular binding pool design, multiple cell types can be separated efficiently at the same time; the annular binding pool is designed with an annular channel, which allows samples to circulate multiple times, significantly improving the cell capture efficiency, especially for low-abundance cells; the piezoelectric micropump is connected to the annular channel, which can extend the flow time of cells in the annular channel and the cleaning time of the cleaning solution in the annular channel;

[0039] 2. The use of antibodies with different expression tags and specific capture antibodies pre-embedded in the separation reaction chamber greatly improves the specificity of cell separation, reduces non-specific binding, and improves the accuracy of analysis results;

[0040] 3. Integrate cell separation, washing, PCR reaction and detection in a microfluidic device to achieve integrated operation of the entire process from sample processing to result analysis, significantly reducing the sample transfer steps and reducing the risk of sample loss and contamination; the integrated design greatly shortens the time from sample processing to result acquisition, improves experimental efficiency, and is conducive to rapid diagnosis and research progress;

[0041] 4. The use of microfluidic control systems achieves highly automated operations, reduces human errors, improves the repeatability and reliability of experiments, simplifies the operation process, and reduces the requirements for the professional skills of operators;

[0042] 5. By using precisely controlled mineral oil isolation, cross contamination in multiple PCR reactions is effectively prevented, ensuring the independence of multiple reaction areas without affecting the PCR reaction;

[0043] 6. In situ PCR and real-time fluorescence detection systems improve the sensitivity of detection, especially for the analysis of rare cells. Real-time monitoring can provide more dynamic information and facilitate more in-depth data analysis;

[0044] 7. Microfluidic design significantly reduces the amount of samples and reagents used, reduces experimental costs, and is particularly suitable for the analysis of precious or rare samples;

[0045] 8. The design of the device allows the number and configuration of separation reaction chambers to be adjusted according to different experimental requirements. It is suitable for various types of cell separation and molecular detection requirements and has broad application prospects. The honeycomb point heating module design allows independent and precise temperature control of multiple separation reaction chambers at the same time, and can easily adjust the number and position of heating areas according to different experimental requirements without changing hardware.

[0046] 9. The high efficiency and precision of this device give it broad application prospects in clinical diagnosis, personalized medicine, drug screening and other fields, and will help promote the development of precision medicine and single-cell analysis technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is an overall schematic diagram of a microfluidic cell separation detection device according to an embodiment of the utility model;

[0048] FIG2 (a) is an exploded schematic diagram of the assembly of a separation unit, a heating unit and a fluorescence detection module according to an embodiment of the present invention;

[0049] FIG2( b ) is a schematic diagram of a separation unit, a heating unit and a fluorescence detection module assembly according to an embodiment of the present invention;

[0050] FIG2 (c) is a top view of the assembled separation unit, heating unit and fluorescence detection module of one embodiment of the present invention;

[0051] Figure 3 It is an enlarged schematic diagram of a heating module according to an embodiment of the present utility model;

[0052] FIG4 (a) is a schematic diagram of a microfluidic chip and a chip fixing frame in a separated state according to an embodiment of the present invention;

[0053] FIG4( b ) is a top view of a microfluidic chip according to an embodiment of the present invention;

[0054] FIG5 (a) is a schematic diagram of a microfluidic chip having three separation chambers in a binding pool according to an example of the present invention;

[0055] FIG5 (b) is a schematic diagram of a microfluidic chip having four separation chambers in a binding pool according to an example of the present invention;

[0056] FIG5( c ) is a schematic diagram of a microfluidic chip having five separation chambers in a binding pool according to an example of the present invention.

[0057] Explanation of the numbers in the figure: 1. Microfluidic chip; 2. Chip fixing frame; 3. Binding pool; 4. Sample addition hole; 5. Cleaning solution addition hole; 6. PCR reaction solution addition hole; 7. Waste liquid discharge hole; 8. Mineral oil addition hole; 9-1. First separation reaction chamber; 9-2. Second separation reaction chamber; 9-3. Third separation reaction chamber; 9-4. Fourth separation reaction chamber; 10. Temperature control system; 11. Fluorescence detection module; 12. Control system; 13. Heating module; 14. Piezoelectric micropump; 15. Chassis; 16. Lock. DETAILED DESCRIPTION

[0058] In order to make the purpose, technical solution and advantages of the implementation of the utility model clearer, the technical solution in the implementation of the utility model will be clearly and completely described below in conjunction with the drawings in the implementation of the utility model. Obviously, the described implementation is a part of the implementation of the utility model, not all of the implementations. Based on the implementation of the utility model, all other implementations obtained by ordinary technicians in this field without creative work are within the scope of protection of the utility model.

[0059] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0060] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.

[0061] In order to better understand the purpose, structure and function of the present invention, a microfluidic cell separation and detection device of the present invention is further described in detail below in conjunction with the accompanying drawings.

[0062] Example 1

[0063] like Figure 1 As shown in FIG. 2 (a), FIG. 2 (b) and FIG. 2 (c), the utility model is a microfluidic cell separation detection device, comprising a chassis 15, on which a separation unit, a heating unit, a control system and a fluorescence detection module 11 are disposed; the heating unit and the fluorescence detection module 11 are both connected to the separation unit and the control system 12;

[0064] The separation unit comprises a microfluidic chip 1 and a chip fixing frame 2, wherein the microfluidic chip 1 is embedded in the chip fixing frame 2; the microfluidic chip 1 is provided with a liquid inlet channel, a binding pool 3 and a liquid outlet channel, wherein the binding pool 3 has a liquid inlet side and a liquid outlet side, wherein the liquid inlet channel has a liquid inlet port, wherein the liquid inlet channel is correspondingly connected to the liquid inlet side of the binding pool, wherein the liquid outlet channel has a waste liquid discharge hole 7, wherein the liquid outlet channel is correspondingly connected to the liquid outlet side of the binding pool 3;

[0065] The heating unit comprises a heating module 13, and the heating module 13 is arranged between the chip fixing frame 2 and the binding pool 3;

[0066] The combination pool 3 is provided with a plurality of separation reaction chambers and mineral oil adding holes 8 which are interconnected.

[0067] As shown in FIG. 4( a ) and FIG. 4( b ), the chip fixing frame 2 is provided with a recess, and the microfluidic chip 1 is fixed in the recess.

[0068] Example 2

[0069] like Figure 1 2(a), 2(b) and 2(c), the utility model is a microfluidic cell separation detection device, comprising a chassis 15, on which a separation unit, a heating unit, a control system and a fluorescence detection module 11 are arranged; the heating unit and the fluorescence detection module 11 are electrically connected to the separation unit and the control system 12;

[0070] The separation unit comprises a microfluidic chip 1 and a chip fixing frame 2, wherein the microfluidic chip 1 is embedded in the chip fixing frame 2; the microfluidic chip 1 is provided with a liquid inlet channel, a binding pool 3 and a liquid outlet channel, wherein the binding pool 3 has a liquid inlet side and a liquid outlet side, wherein the liquid inlet channel has a liquid inlet port, wherein the liquid inlet channel is correspondingly connected to the liquid inlet side of the binding pool, wherein the liquid outlet channel has a waste liquid discharge hole 7, wherein the liquid outlet channel is correspondingly connected to the liquid outlet side of the binding pool 3;

[0071] The heating unit comprises a heating module 13, and the heating module 13 is arranged between the chip fixing frame 2 and the binding pool 3;

[0072] The combination pool 3 is provided with a plurality of separation reaction chambers and mineral oil adding holes 8 which are interconnected.

[0073] The difference between this embodiment and embodiment 1 is that:

[0074] As shown in FIG. 4 (a) and FIG. 4 (b), the binding pool 3 may be designed with a spiral, serpentine or tree-like branching structure, or may be designed as a multi-level binding pool structure to achieve a more complex separation strategy; Figure 1 As shown, in this embodiment, the binding pool 3 is annular, and the liquid inlet channel includes a sample liquid inlet channel, a cleaning liquid liquid inlet channel and a PCR reaction liquid liquid inlet channel; the sample liquid inlet channel has a sample addition hole 4, the cleaning liquid liquid inlet channel has a cleaning liquid addition hole 5, and the PCR reaction liquid liquid inlet channel has a PCR reaction liquid addition hole 6; the sample liquid inlet channel, the cleaning liquid liquid inlet channel and the PCR reaction liquid liquid inlet channel are connected to the liquid inlet side of the annular binding pool 3 after merging;

[0075] The sample addition hole 4, the cleaning solution addition hole 5, the PCR reaction solution addition hole 6 and the waste liquid discharge hole 7 are all provided with valves correspondingly for controlling the opening and closing of the sample addition hole 4, the cleaning solution addition hole 5, the PCR reaction solution addition hole 6 and the waste liquid discharge hole 7;

[0076] In addition to using PCR reaction solutions, you can also consider using isothermal amplification techniques such as LAMP (loop-mediated isothermal amplification) or RPA (recombinase polymerase amplification);

[0077] In addition to using mineral oil for isolation, other immiscible liquids such as fluorocarbon liquids can also be considered for isolation; physical partitions or controllable valve systems can also be designed for reaction isolation;

[0078] The binding pool 3 has an annular channel, one side of which is connected to the liquid inlet channel, and the other side of which is connected to the liquid outlet channel; a plurality of separation reaction chambers are arranged on the annular channel, and a mineral oil addition hole 8 is arranged on the inner side of the annular channel, and the mineral oil addition hole 8 is connected to a plurality of delivery channels connected to the annular channel. The design of the annular channel allows the sample to circulate multiple times, which significantly improves the cell capture efficiency, especially for low-abundance cells; as shown in FIG5 , different numbers of separation reaction chambers can be arranged on the annular channel of the binding pool 3 as needed. 5 (a) is a combined pool 3 with three separation reaction chambers, FIG. 5 (b) is a combined pool 3 with four separation reaction chambers, and FIG. 5 (c) is a combined pool 3 with five separation reaction chambers. In this embodiment, the annular channel is provided with an average of four separation reaction chambers, namely, a first separation reaction chamber 9-1, a second separation reaction chamber 9-2, a third separation reaction chamber 9-3, and a fourth separation reaction chamber 9-4. The mineral oil adding hole 8 is connected with four delivery channels corresponding to the four separation reaction chambers, and the delivery channels are connected with the annular channel.

[0079] A piezoelectric micropump 14 is also connected to the annular channel, and the piezoelectric micropump 14 is electrically connected to the control system 12. After the sample cells are transported to the binding pool 3, the piezoelectric micropump 14 is used to prolong the flow time of the cells in the annular channel of the binding pool, so that the cells and antibodies can be better combined. Specifically, the piezoelectric micropump 14 is used to control the sample to circulate in the binding pool 3 at a speed of 20 μL / min for 15 minutes; when the cleaning solution is transported to the binding pool 3, the piezoelectric micropump 14 is used to prolong the flow time of the cleaning solution in the annular channel of the binding pool 3, thereby prolonging the cleaning time and making the channel clean more thoroughly.

[0080] Example 3

[0081] like Figure 1 2(a), 2(b) and 2(c), the utility model is a microfluidic cell separation detection device, comprising a chassis 15, on which a separation unit, a heating unit, a control system and a fluorescence detection module 11 are arranged; the heating unit and the fluorescence detection module 11 are electrically connected to the separation unit and the control system 12;

[0082] The separation unit comprises a microfluidic chip 1 and a chip fixing frame 2, wherein the microfluidic chip 1 is embedded in the chip fixing frame 2; the microfluidic chip 1 is provided with a liquid inlet channel, a binding pool 3 and a liquid outlet channel, wherein the binding pool 3 has a liquid inlet side and a liquid outlet side, wherein the liquid inlet channel has a liquid inlet port, wherein the liquid inlet channel is correspondingly connected to the liquid inlet side of the binding pool, wherein the liquid outlet channel has a waste liquid discharge hole 7, wherein the liquid outlet channel is correspondingly connected to the liquid outlet side of the binding pool 3;

[0083] The heating unit comprises a heating module 13, and the heating module 13 is arranged between the chip fixing frame 2 and the binding pool 3;

[0084] The combination pool 3 is provided with a plurality of separation reaction chambers and mineral oil adding holes 8 which are interconnected.

[0085] The difference between this embodiment and the above embodiments is that:

[0086] like Figure 1 2(a), 2(b) and 2(c), the heating unit further comprises a temperature control system 10, the temperature control system 10 is connected to the heating module 13, the temperature control system 10 is arranged in the chassis 15, and the temperature control system 10 is also electrically connected to the control system 12, for accurately controlling the temperature of the heating module 13;

[0087] like Figure 3 As shown, the heating module 13 is in a honeycomb dot shape, allowing independent and precise temperature control of each separate reaction chamber, and the number and position of the heating areas can be easily adjusted according to different experimental requirements without changing the hardware;

[0088] The chassis 15 is also provided with a lock 16 that cooperates with the fluorescence detection module 11. The fluorescence detection module 11 is fixed to the binding pool 3 through the lock 16 to monitor the fluorescence signals of each separation chamber in real time. Multiple devices can also be used together to facilitate the separation and detection of multiple cells at the same time.

[0089] As shown in Figure 2 (a), Figure 2 (b), Figure 2 (c), Figure 3 As shown in FIG. 4 (a) and FIG. 4 (b), the utility model is a microfluidic cell separation detection device, and its use process is now described in detail by taking examples:

[0090] Step 1: Sample pretreatment;

[0091] Prepare four antibodies with different expression tags:

[0092] Anti-CD4-His tag antibody (for CD4+ T cells),

[0093] Anti-CD8-Flag antibody (for CD8+ T cells),

[0094] Anti-CD19-HA tag antibody (for B cells),

[0095] Anti-CD56-Myc tag antibody (for NK cells);

[0096] The four antibodies were mixed with PBMC samples after lysing red blood cells and incubated at 4°C for 30 minutes.

[0097] Step 2: Chip preparation and sample loading;

[0098] like Figure 1 As shown, four different regions of the annular binding pool 3 are pre-embedded respectively:

[0099] Anti-His tag antibody (Region A first separation reaction chamber 9-1, used to capture CD4+ T cells),

[0100] Anti-Flag tag antibody (area B second separation reaction chamber 9-2, used to capture CD8+ T cells),

[0101] Anti-HA tag antibody (region C third separation reaction chamber 9-3, used to capture B cells),

[0102] Anti-Myc tag antibody (region D fourth separation reaction chamber 9-4, used to capture NK cells);

[0103] The pretreated PBMC sample is injected into the annular binding pool 3 through the sample addition hole 4; the piezoelectric micropump 14 is started to control the sample to circulate in the binding pool at a speed of 20 μL / min for 15 minutes;

[0104] During this process, CD4+ T cells, CD8+ T cells, B cells, and NK cells were captured in the corresponding area A, B, C, and D chambers, respectively.

[0105] Step 3: Cleaning;

[0106] PBS buffer is injected through the cleaning solution addition hole 5 for cleaning, and then unbound cells and impurities are discharged through the waste liquid discharge hole 7; the first two steps are repeated three times to ensure sufficient cleaning.

[0107] Step 4: PCR reaction preparation;

[0108] Inject specific PCR reaction mixture into the chambers of regions A, B, C, and D through the PCR reaction solution adding hole 6:

[0109] CD4 gene specific primer and probe mixture (Region A chamber),

[0110] CD8 gene specific primer and probe mixture (chamber area B),

[0111] CD19 gene specific primer and probe mixture (chamber area C),

[0112] CD56 gene-specific primer and probe mixture (region D chamber);

[0113] Step 5: Reaction isolation;

[0114] A small amount of mineral oil is accurately injected into the annular channel through the mineral oil injection hole 8 to form an isolation zone.

[0115] Step 6: PCR reaction and detection;

[0116] Start the honeycomb point heating module 13, which is designed to be located on the chip fixing frame 2, and can accurately control the temperature of the four separation reaction chambers on the microfluidic chip 1 to perform the following PCR program:

[0117] 95°C for 3 minutes,

[0118] 95°C for 15 seconds, 60°C for 60 seconds (40 cycles),

[0119] During the 60°C stage of each cycle, the fluorescence detection module 11 is used to monitor the fluorescence signal of each area in real time.

[0120] Step 7: Data analysis;

[0121] The control system 12 collects the fluorescence signal data, draws an amplification curve, and then calculates the threshold cycle number (Ct value) of the amplification curve.

[0122] The above description is only the preferred embodiment of the utility model, and is not intended to limit the utility model. For those skilled in the art, the utility model can have various modifications and changes. In the absence of contradiction, the various embodiments can be combined with each other to form a new embodiment. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the utility model are included in the protection scope of the utility model.

Claims

1. A microfluidic cell separation detection device, characterized in that: It comprises a chassis, on which a separation unit, a heating unit, a control system and a fluorescence detection module are arranged; the heating unit and the fluorescence detection module are both electrically connected to the separation unit and the control system; The separation unit comprises a microfluidic chip and a chip fixing frame, wherein the microfluidic chip is embedded in the chip fixing frame; the microfluidic chip is provided with a liquid inlet channel, a binding pool and a liquid outlet channel, wherein the binding pool has a liquid inlet side and a liquid outlet side, wherein the liquid inlet channel has a liquid inlet port, wherein the liquid inlet channel is correspondingly connected to the liquid inlet side of the binding pool, wherein the liquid outlet channel has a waste liquid discharge hole, wherein the liquid outlet channel is correspondingly connected to the liquid outlet side of the binding pool; The heating unit comprises a heating module, and the heating module is arranged between the chip fixing frame and the binding pool; The combination pool is provided with a plurality of separation reaction chambers and mineral oil adding holes which are interconnected.

2. The microfluidic cell separation detection device according to claim 1, characterized in that: The liquid inlet channel includes a sample liquid inlet channel, a cleaning liquid inlet channel and a PCR reaction liquid inlet channel; the sample liquid inlet channel has a sample addition hole, the cleaning liquid inlet channel has a cleaning liquid addition hole, and the PCR reaction liquid inlet channel has a PCR reaction liquid addition hole; The sample inlet channel, the cleaning solution inlet channel and the PCR reaction solution inlet channel are connected to the inlet side of the binding pool.

3. The microfluidic cell separation detection device according to claim 2, characterized in that: The sample adding hole, the cleaning solution adding hole, the PCR reaction solution adding hole and the waste liquid discharging hole are all provided with valves accordingly.

4. The microfluidic cell separation detection device according to claim 1, characterized in that: The heating unit further comprises a temperature control system, the temperature control system is connected to the heating module, the temperature control system is arranged in the chassis, and the temperature control system is also electrically connected to the control system.

5. The microfluidic cell separation detection device according to claim 1, characterized in that: The chassis is also provided with a lock buckle that cooperates with the fluorescence detection module.

6. The microfluidic cell separation detection device according to any one of claims 1 to 5, characterized in that: The binding pool is annular.

7. The microfluidic cell separation detection device according to claim 6, characterized in that: The combining pool has an annular channel, one side of the annular channel is communicated with the liquid inlet channel, and the other side of the annular channel is communicated with the liquid outlet channel.

8. The microfluidic cell separation detection device according to claim 7, characterized in that: The annular channel is also connected with a piezoelectric micro pump, and the piezoelectric micro pump is electrically connected to the control system.

9. The microfluidic cell separation detection device according to claim 7, characterized in that: A plurality of separation reaction chambers are arranged on the annular channel, a mineral oil adding hole is arranged on the inner side of the annular channel, and the mineral oil adding hole is connected to a plurality of conveying channels communicating with the annular channel.

10. The microfluidic cell separation detection device according to claim 1, characterized in that: The heating module is in the shape of a honeycomb point.

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