A microfluidic chip

Abstract

The utility model provides a kind of microfluidic chip, including the mixing layer, dilution layer, filter layer and focusing layer that are sequentially stacked, each functional layer internal flow passage sequence communication, to build a compact structure, complete automation cell sample processing system of function.Firstly, mixed flow channel is used to mix cell liquid and diluent, then through mixing and diluting cavity, cell liquid is efficiently and uniformly gradient diluted;Subsequently, sample flow is filtered to accurately remove impurities and cell mass, improve the purity of sample.Finally, the liquid focusing section in focusing layer can arrange the processed cells by hydrodynamics, so that it is arranged in order to pass, to create ideal conditions for subsequent cell counting device accurate optical detection and counting.By sequentially stacking the four functional layers of mixing, dilution, filtration and focusing, the overall volume of the microfluidic device is significantly reduced, and the automation and optimization of the whole sample processing process are realized.

Description

Technical Field

[0001] This utility model relates to the field of cell encapsulation technology, and in particular to a microfluidic chip. Background Technology

[0002] With the rapid development of biomedical technology, single-cell analysis technology is playing an increasingly important role in disease diagnosis, drug development, and biological research. Currently, commonly used cell analysis equipment mainly includes flow cytometers, one of whose core technologies is the realization of single-cell analysis and precise detection. Traditional flow cytometers use a pressure-driven sheath flow focusing method, which can achieve single-cell analysis, but the equipment is bulky, expensive, and requires professional operation. Furthermore, existing technologies cannot simultaneously accommodate cells of different sizes, limiting the application range of the equipment. Utility Model Content

[0003] This invention provides a microfluidic chip that integrates mixing, dilution, filtration and focusing functions, achieving a high degree of integration in sample processing and reducing the size of microfluidic devices.

[0004] This utility model provides a microfluidic chip, comprising: a mixing layer, a dilution layer, a filter layer, and a focusing layer; the dilution layer, the filter layer, and the focusing layer are sequentially stacked on the mixing layer, and the mixing layer is disposed in the chip shell;

[0005] The mixing layer includes a mixing channel, a cell fluid inlet, a diluent inlet, and a waste liquid outlet; the dilution layer includes a liquid inlet and a mixing and dilution chamber; the focusing layer includes a cell fluid accommodating chamber, a liquid focusing section, and a waste liquid recovery channel.

[0006] Cell fluid and diluent flow into the mixing channel through the cell fluid inlet and the diluent inlet, respectively. The mixing channel, the liquid inlet, the mixing and dilution chamber, the filter layer, the cell fluid container chamber, and the liquid focusing section are connected in sequence. The liquid focusing section is used to organize the cells in the cell mixture so that they are arranged in order to facilitate focusing and imaging by the cell counting device.

[0007] The liquid focusing section is connected to the waste liquid recovery channel; the waste liquid is discharged through the waste liquid outlet after passing through the waste liquid recovery channel.

[0008] Furthermore, the mixing channel includes multiple mixing units, and the mixing units are connected to each other via U-shaped channels;

[0009] The mixing unit includes multiple mixing chambers connected in sequence.

[0010] The shape of the mixing cavity is at least one of rhombus, Tesla valve shape and concentric circle.

[0011] Furthermore, the mixing and dilution chamber is provided with a plurality of first microcolumn structures; the cell fluid containing chamber is provided with a plurality of second microcolumn structures;

[0012] Multiple second micropillar structures are arranged in a one-to-one correspondence with multiple first micropillar structures, and the filter layer is located between multiple first micropillar structures and multiple second micropillar structures.

[0013] Furthermore, the liquid focusing segment includes: a first liquid focusing segment and a second liquid focusing segment;

[0014] The first liquid focusing section includes multiple U-shaped bends connected end to end.

[0015] The inlet of the first U-shaped curved flow channel is connected to the cell fluid accommodating cavity;

[0016] The outlet of the last curved flow channel is connected to the inlet of the second liquid focusing section.

[0017] Furthermore, the second liquid focusing segment is linear.

[0018] Furthermore, the mixing layer also includes a waste liquid output channel; the focusing layer also includes a drain hole;

[0019] The input ends of at least two of the waste liquid recovery channels are connected to the output end of the second liquid focusing section. After the output ends converge, they are connected to the drain hole through the liquid outlet. The drain hole is connected to the waste liquid output channel, and the waste liquid output channel is connected to the waste liquid outlet.

[0020] Furthermore, the chip casing is provided with a recessed mounting platform, and the filter layer is disposed within the mounting platform, located between the dilution layer and the focusing layer.

[0021] Furthermore, the chip casing is also provided with a viewing window, the position of which corresponds to the focusing channel of the focusing layer, for optical detection of cells.

[0022] Furthermore, the mixing layer, the dilution layer, the filter layer, and the focusing layer are connected by bonding.

[0023] Furthermore, it also includes guide grooves and alignment holes;

[0024] The guide groove is disposed on the side wall of the chip housing and is used to cooperate with the guide structure of the cell counting device;

[0025] The alignment wells sequentially penetrate the mixing layer, the dilution layer, and the focusing layer, and are used to align and fix the cell counting device.

[0026] Compared with the prior art, the present invention has at least the following technical effects:

[0027] The microfluidic chip disclosed in this invention comprises a mixing layer, a dilution layer, a filtration layer, and a focusing layer stacked sequentially. The internal channels of each functional layer are sequentially connected, thus constructing a compact and fully functional automated cell sample processing system. First, the mixing channel mixes the cell solution and diluent. Then, the mixing and dilution chamber performs efficient and uniform gradient dilution of the cell solution. Subsequently, the sample flows through the filtration layer to precisely remove impurities and cell clumps, improving sample purity. Finally, the liquid focusing section in the focusing layer hydrodynamically organizes the processed cells, ensuring they pass through in an orderly manner, creating ideal conditions for precise optical detection and counting in subsequent cell counting devices. By sequentially stacking and integrating these four functional layers (mixing, dilution, filtration, and focusing), the overall size of the microfluidic device is significantly reduced, and the entire sample processing process is automated and optimized. Attached Figure Description

[0028] Figure 1 This is a simplified schematic diagram of the microfluidic chip structure in one embodiment of this utility model;

[0029] Figure 2 This is a simplified schematic diagram of another structure of the microfluidic chip in one embodiment of this utility model;

[0030] Figure 3 This is a simplified schematic diagram of the mixing layer structure in one embodiment of this utility model;

[0031] Figure 4 This is an enlarged view of the mixing channel in one embodiment of the present invention;

[0032] Figure 5 This is a simplified schematic diagram of the dilution layer structure in one embodiment of this utility model;

[0033] Figure 6 This is a simplified schematic diagram of the focusing layer structure in one embodiment of the present invention;

[0034] Figure 7 This is one embodiment of the present invention. Figure 6 Enlarged view of the structure at point A in the middle.

[0035] Reference numerals: 5. Mixing layer; 6. Dilution layer; 7. Filtering layer; 8. Focusing layer; 1. Waste liquid outlet; 2. Cell fluid inlet; 3. Dilution liquid inlet; 55. Mixing channel outlet; 54. First channel; 53. Second channel; 52. Mixing channel; 522. Mixing chamber; 521. U-shaped channel; 62. Mounting platform; 61. Liquid inlet; 64. Drain hole; 65. First microcolumn structure; 66. Mixing and dilution chamber; 81. Cell fluid accommodating chamber; 82. Second microcolumn structure; 86. Liquid outlet; 83. First liquid focusing section; 84. Second liquid focusing section; 57. Waste liquid outlet channel; 85. Waste liquid recovery channel; 59. Viewing window; 58. Alignment hole; 500. Guide groove. Detailed Implementation

[0036] The following description, in conjunction with schematic diagrams, illustrates a microfluidic chip according to the present invention, showing preferred embodiments of the invention. It should be understood that those skilled in the art can modify the invention described herein while still achieving its advantageous effects. Therefore, the following description should be understood as being of general knowledge to those skilled in the art and is not intended to limit the scope of the invention.

[0037] The present invention will be described more specifically by way of example with reference to the accompanying drawings in the following paragraphs. The advantages and features of the present invention will become clearer from the following description. It should be noted that the drawings are in a very simplified form and use non-precise proportions, and are only used to facilitate and clarify the illustration of the embodiments of the present invention.

[0038] Please refer to Figures 1-7 This invention provides a microfluidic chip, comprising a mixing layer 5, a dilution layer 6, a filter layer 7, and a focusing layer 8 stacked sequentially. The dilution layer 6, the filter layer 7, and the focusing layer 8 are stacked sequentially on the mixing layer 5, which is disposed within the chip casing.

[0039] The mixing layer 5 includes a mixing channel 52, a cell fluid inlet 2, a diluent inlet 3, and a waste liquid outlet 53; the dilution layer 6 includes a liquid inlet 61 and a mixing and dilution chamber 66; the focusing layer 8 includes a cell fluid accommodating chamber 81, a liquid focusing section, and a waste liquid recovery channel 85.

[0040] Cell fluid and diluent flow into the mixing channel 52 through the cell fluid inlet 2 and the diluent inlet 3, respectively. The mixing channel 52, the liquid inlet 61, the mixing and dilution chamber 66, the filter layer 7, the cell fluid container chamber 81, and the liquid focusing section are connected in sequence. The liquid focusing section is used to organize the cells in the cell mixture so that they are arranged in order to facilitate focusing and imaging by the cell counting device.

[0041] The liquid focusing section is connected to the waste liquid recovery channel 85; the waste liquid is discharged through the waste liquid outlet 53 after passing through the waste liquid recovery channel 85.

[0042] The microfluidic chip disclosed in this invention includes a mixing layer 5, a dilution layer 6, a filtration layer 7, and a focusing layer 8 stacked sequentially. The internal channels of each functional layer are sequentially connected, thus constructing a compact and fully functional automated cell sample processing system. First, the mixing channel 52 mixes the cell solution and diluent. Then, the mixing and dilution chamber 66 performs efficient and uniform gradient dilution of the cell solution. Subsequently, the sample flows through the filtration layer 7 to precisely remove impurities and cell clumps, improving sample purity. Finally, the liquid focusing section in the focusing layer 8 hydrodynamically organizes the processed cells, ensuring they pass through in an orderly manner, creating ideal conditions for precise optical detection and counting in subsequent cell counting devices. By sequentially stacking and integrating these four functional layers (mixing, dilution, filtration, and focusing), the overall volume of the microfluidic device is significantly reduced, and the entire sample processing process is automated and optimized.

[0043] Please refer to Figure 3 In one specific embodiment, the mixing layer 5 includes a first channel 54 and a second channel 53.

[0044] Specifically, the cell fluid inlet 2 and the diluent inlet 3 are respectively connected to the inlets of the first channel 54 and the second channel 53; the outlets of the first channel 54 and the second channel 53 are both connected to the inlet of the mixing channel.

[0045] In this embodiment, cell slurry dilution is achieved by precisely controlling the flow rate ratio of cell slurry and diluent. Specifically, two independent peristaltic pumps or syringe pumps are used to control the input of cell slurry and diluent, respectively. In one specific embodiment, cell slurry is pumped in through cell slurry inlet 2, and diluent is pumped in through diluent inlet 3. The dilution factor is controlled by adjusting the flow rate ratio of the two pumps. For example, when it is necessary to dilute the cell slurry 10 times, the flow rate of the diluent pump can be set to 9 times that of the cell slurry pump. After the two liquids are initially mixed in the mixing channel, they enter the mixing and dilution chamber 66 for thorough mixing. The cell concentration in the mixed liquid will be reduced to 1 / 10 of the original concentration. In this way, the operator can achieve precise dilution of different factors by simply adjusting the flow rate ratio of the two pumps according to experimental needs. The dilution factor can be continuously adjusted within a wide range, and the dilution process is stable and controllable.

[0046] In one specific embodiment, the cell fluid inlet 2 and the diluent inlet 3 are respectively located on cylindrical protrusions. The top of the protrusion has a through hole for the introduction or export of liquid, and the bottom of the protrusion smoothly transitions to the chip body. Furthermore, a rotating locking sleeve is provided on the top of the protrusion, enabling quick connection and sealing with external pipelines. It is understood that those skilled in the art can design cell fluid inlets 2 and diluent inlets 3 with different structures according to actual conditions, and no specific limitations are made here.

[0047] In another specific embodiment, the cell fluid inlet 2 and the diluent inlet 3 are respectively located on one side edge of the mixing layer 5 shell, and a connection gap is provided between them to ensure that the pipelines do not interfere with each other when connected.

[0048] Please refer to Figure 4 In this embodiment, the mixing channel 52 includes multiple mixing units, and the mixing units are connected to each other through a U-shaped channel 521; each mixing unit includes multiple mixing chambers connected in sequence.

[0049] Specifically, after the cell sap and diluent enter the mixing channel 52, they first flow through multiple mixing chambers 522 within the first mixing unit. The continuous arrangement of the mixing chambers 522 causes the fluid to undergo multiple divisions and recombinations during the flow, achieving initial mixing in a laminar flow state. Subsequently, the fluid enters the next mixing unit through the U-shaped channel 521. The tortuous structure of the U-shaped channel 521 induces secondary flow in the fluid, further breaking the laminar interface.

[0050] In this embodiment, the solution combines a mixing unit with a U-shaped flow channel 521, which retains the advantage of low shear force while actively inducing fluid disturbance through geometric structure. This enables efficient mixing within a shorter flow channel length, while avoiding mechanical damage to cells caused by high-speed flow.

[0051] In another specific embodiment, the shape of the mixing unit is at least one of rhombus, Tesla valve shape, and concentric circle.

[0052] Specifically, the rhomboid mixing chamber generates a vortex effect through its sudden expansion and contraction structure, enhancing the mixing effect. The Tesla valve-shaped mixing chamber utilizes a valveless structure to generate directional vortices, improving mixing efficiency. The concentric circular mixing chamber induces swirling flow in the fluid, strengthening radial mixing. Those skilled in the art can choose the structure of the mixing chamber according to the actual situation.

[0053] In another specific embodiment, the corner of the U-shaped flow channel 521 adopts a rounded transition design to reduce the resistance loss of the liquid during the flow process.

[0054] Please continue to refer to this. Figure 5In this embodiment, the mixing and dilution chamber 66 is provided with a plurality of first micropillar structures 65.

[0055] In one specific embodiment, a plurality of the first micropillar structures 65 are arranged in a lattice, with equal distances between each pair of the first micropillar structures 65. This equidistant arrangement ensures uniform fluid resistance among the first micropillar structures 65, thereby making the flow velocity and residence time within each first micropillar structure 65 essentially the same.

[0056] In another specific embodiment, a plurality of first micropillar structures 65 are arranged in a 7×3 lattice structure.

[0057] Furthermore, the cell fluid accommodating cavity 81 is provided with a plurality of second micropillar structures 82, and the plurality of second micropillar structures 82 are provided in a one-to-one correspondence with a plurality of first micropillar structures 65. The filter layer 7 is located between the plurality of first micropillar structures 65 and the plurality of second micropillar structures 82.

[0058] Please refer to Figure 2 and Figure 5 In this embodiment, the chip casing is provided with a recessed mounting platform 62, and the filter layer 7 is disposed in the mounting platform 62, located between the dilution layer 6 and the focusing layer 8.

[0059] Furthermore, in this embodiment, a porous membrane is provided within the filter layer 7. In a specific embodiment, a porous membrane material with a specific pore size can be selected, such as a polycarbonate membrane, a polyester membrane, or a silicon-based porous membrane. The advantage of selecting the above-mentioned porous membrane materials is that materials such as polycarbonate membranes, polyester membranes, and silicon-based porous membranes have excellent chemical stability and mechanical strength, can maintain stable filtration performance under various working conditions, and also have good biocompatibility.

[0060] Understandably, the pore size of the filter membrane is selected based on the size of the target cells. The selection is based on the principle of trapping large particles and cell aggregates so that only individual cells that meet the size requirements can pass through the filter layer and enter the subsequent focusing channel.

[0061] In one specific embodiment, for cells ranging from 5 μm to 25 μm, the pore size of the filter membrane is typically set to 30 μm to 50 μm to effectively trap particles and cell aggregates larger than the target cell size, while allowing individual target cells to pass through smoothly, thereby improving the normal operation of the subsequent focusing channel and the accuracy of cell counting.

[0062] Furthermore, in this embodiment, the liquid focusing segment includes: a first liquid focusing segment 83 and a second liquid focusing segment 84.

[0063] Please refer to Figure 7Specifically, the first liquid focusing section 83 includes multiple U-shaped curved channels connected end to end. The inlet of the first U-shaped curved channel is connected to the cell fluid receiving cavity 81. The outlet of the last curved channel is connected to the inlet of the second liquid focusing section 84.

[0064] In this embodiment, the first liquid focusing section 83 is provided with multiple U-shaped curved channels. The liquid is driven by external power to flow at high speed in the U-shaped curved channels. Due to the inertial effect, a secondary flow is generated. This secondary flow will generate a lateral force on the cells, causing the cells to gradually gather towards the liquid focusing section. The first liquid focusing section 83 is used to achieve the final focusing and positioning of the cells under a stable flow rate, so that the cells move in a single row along the second liquid focusing section 84. Compared with other focusing methods that require additional sound fields, electric fields or magnetic fields, this hydrodynamic focusing method can achieve cell focusing only by relying on the physical properties of the fluid itself. It has the characteristics of simple structure, convenient operation and easy control.

[0065] It is understood that the size and number of the U-shaped curved channels are configured according to the different flow rates of the cell mixture and the size of the cells, and no specific limitations are imposed here. Specifically, the selection of parameters for the U-shaped curved channels follows these rules: the channel size is directly proportional to the target particle size, that is, the smaller the particles, the more proportionally the channel depth and width need to be reduced; the number of channels is inversely proportional to the particle size, that is, the smaller the particles, the more U-shaped curved channels are required.

[0066] In one specific embodiment, for cells with a size of 5um to 25um, such as NK (natural killer) cells, CHO (Chinese hamster ovary) cells, etc., the depth of the U-shaped curved channel is 30-40um, the width is 75-120um, and the number of U-shaped curved channels is more than 5.

[0067] In another specific embodiment, when processing smaller particles, such as platelets or certain large bacteria with sizes ranging from 2 μm to 10 μm, the parameters of the focusing channel also need to be optimized accordingly. To generate effective inertial focusing force for these smaller particles, the channel size needs to be reduced proportionally. In this case, the depth of the U-shaped bend channel can be designed to be 15-25 μm, and the width is correspondingly narrowed to 40-60 μm. Simultaneously, to ensure that these tiny particles can achieve sufficient lateral migration in the fluid, the number of U-shaped bend channels typically needs to be increased, for example, to eight or more, to accumulate more secondary flow effects, thereby achieving effective initial aggregation before entering the straight focusing section.

[0068] Furthermore, in this embodiment, the second liquid focusing segment 84 is linear.

[0069] Specifically, the advantage of setting a straight second liquid focusing section 84 is that after the cells have initially aggregated in the U-shaped bend at the front end, they have been roughly pushed towards the center of the channel. At this point, the straight design avoids complex secondary flows caused by the bend, thus stabilizing the flow velocity. In this stable flow field, the initially aggregated cells can be precisely positioned on the centerline of the channel, forming a single, equally spaced column.

[0070] Furthermore, in this embodiment, the mixing layer 5 includes a waste liquid output channel 57 and a waste liquid output port 1; the focusing layer 8 also includes a drain hole 64.

[0071] The input end of the waste liquid recovery channel 85 is connected to the output end of the second liquid focusing section 84. After the output end converges, it is connected to the drain hole 64 through the liquid outlet 86. The drain hole 64 is connected to the waste liquid output channel 57, and the waste liquid output channel 57 is connected to the waste liquid output port 1.

[0072] Specifically, after the cell inertial focusing is completed in the focusing layer 8, the unused mixed liquid enters the end of the second liquid focusing section 84 and is then guided to the waste liquid recovery channel 85. The waste liquid recovery channel 85 delivers the liquid to the drain hole 64, which vertically transfers the liquid to the waste liquid output channel 57, and finally discharges the chip through the waste liquid output port 1. The entire waste liquid path adopts a hierarchical through-flow design to avoid liquid stagnation in a single plane.

[0073] In one specific embodiment, the waste liquid recovery channels 85 can be 2, 3, or 5, etc. Multiple waste liquid recovery channels 85 converge and connect to the waste liquid outlet 1.

[0074] Specifically, the advantages of setting up multiple waste liquid recovery channels 85 are as follows: First, multiple waste liquid recovery channels 85 can effectively disperse the waste liquid flow, avoiding back pressure caused by excessive flow in a single channel, thereby improving the stability of liquid flow within the chip; second, when one waste liquid recovery channel 85 becomes blocked, the other waste liquid recovery channels 85 can still operate normally, improving the reliability and robustness of the system; third, the parallel drainage method of multiple waste liquid recovery channels 85 can accelerate the waste liquid drainage speed and improve the overall detection efficiency, which is particularly suitable for high-throughput cell detection applications; in addition, the converging design of multiple channels also helps to balance the fluid resistance of each focusing channel, making the flow velocity of each path in the multi-channel focusing system more uniform, thereby ensuring the consistency of cell focusing effect.

[0075] In another specific embodiment, the waste liquid output channel 57 is W-shaped and is connected to two waste liquid output ports 1.

[0076] In another specific embodiment, each waste liquid outlet 1 has a cylindrical boss structure with a through hole at the top for liquid introduction or export, and the bottom of the boss smoothly transitions to the chip body. Furthermore, a rotating locking sleeve is provided at the top of the boss, enabling quick connection and sealing with external pipelines. It is understood that those skilled in the art can design waste liquid outlet channels with different structures according to actual conditions, and no specific limitations are made here.

[0077] In another specific embodiment, the cell fluid inlet 2 and the diluent inlet 3 are respectively located on one side edge of the mixing layer 5 shell, respectively located on both sides of the cell fluid inlet 2 and the diluent inlet 3, and respectively provided with connection gaps between them to ensure that the pipelines do not interfere with each other when connected.

[0078] In this embodiment, to balance cell focusing effect and cell viability protection, the flow channel design needs to comprehensively consider the relationship between flow rate and shear force. Although a higher flow rate results in better focusing performance, cells can only tolerate a limited amount of shear force. Therefore, the flow rate at the center of the flow channel is generally kept below 1.2 m / s to avoid damaging the cells. Under this flow rate limitation, the system still needs to achieve a high detection throughput. For example, when the center flow rate is 1.2 m / s and the target is a 15 μm cell, the theoretical maximum counting throughput can reach 80,000 cells / s.

[0079] To meet the pressure requirements of high flow rate and high throughput detection, in this embodiment, the mixing layer 5, the dilution layer 6, the filter layer 7 and the focusing layer 8 are connected by bonding.

[0080] Through bonding, the overall chip withstand pressure can reach over 0.5 MPa, meeting the pressure requirements of high flow rate and high throughput detection.

[0081] In one specific embodiment, the bonding connection includes thermocompression bonding, ultrasonic bonding, etc., and no specific limitation is made here. Those skilled in the art can choose different bonding connection methods according to the actual situation, and no specific limitation is made here.

[0082] Furthermore, in this embodiment, the chip casing is also provided with a viewing window 59, the position of which corresponds to the focusing channel of the focusing layer 8, for optical detection of cells. When the chip is used in conjunction with a cell counter, the laser beam can pass through the viewing window 59 and irradiate the focused cell stream, thereby achieving optical detection and counting of cells.

[0083] In one specific embodiment, the material of the viewing window 59 may be optical glass, quartz glass, or optical-grade PMMA, which have good light transmittance, so as to enable accurate transmission of laser signals and improve the accuracy of cell detection.

[0084] Furthermore, in this embodiment, it includes a guide groove 500 and an alignment hole 58;

[0085] Specifically, the guide groove 500 is disposed on the side wall of the mixing layer 5 and is used to cooperate with the guide structure of the cell counting device.

[0086] The alignment hole 58 passes through the mixing layer 5, the dilution layer 6, the filter layer 7 and the focusing layer 8 in sequence, and is used for alignment and fixation in conjunction with the cell counting device.

[0087] In one specific embodiment, the shape of the guide groove 500 can be a dovetail groove, a T-shaped groove, an arc groove, or a stepped protrusion, etc., and its size matches the corresponding guide structure on the cell counter.

[0088] In another specific embodiment, the cell counter can be stably connected to the alignment hole 58 by means of threaded connection, snap-fit ​​fixation, clamping or magnetic adsorption, so as to ensure accurate alignment and reliable fixation between the detection device and the chip.

[0089] In this embodiment, the cell counter is used in conjunction with the microfluidic chip. It achieves precise alignment and fixation with the chip through the alignment hole 58 and guide groove 500 on the chip, counts the cells passing through the focusing channel, and can identify the size, number and characteristics of the cells, thereby achieving accurate cell detection.

[0090] Obviously, those skilled in the art can make various modifications and variations to this utility model without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this utility model and their equivalents, this utility model also intends to include these modifications and variations.

Claims

1. A microfluidic chip, characterized in that, include: A mixing layer, a dilution layer, a filter layer, and a focusing layer are provided; the dilution layer, the filter layer, and the focusing layer are sequentially stacked on the mixing layer, and the mixing layer is disposed in the chip casing; The mixing layer includes a mixing channel, a cell fluid inlet, a diluent inlet, and a waste liquid outlet; the dilution layer includes a liquid inlet and a mixing and dilution chamber; the focusing layer includes a cell fluid accommodating chamber, a liquid focusing section, and a waste liquid recovery channel. Cell fluid and diluent flow into the mixing channel through the cell fluid inlet and the diluent inlet, respectively. The mixing channel, the liquid inlet, the mixing and dilution chamber, the filter layer, the cell fluid container chamber, and the liquid focusing section are connected in sequence. The liquid focusing section is used to organize the cells in the cell mixture so that they are arranged in order to facilitate focusing and imaging by the cell counting device. The liquid focusing section is connected to the waste liquid recovery channel; the waste liquid is discharged through the waste liquid outlet after passing through the waste liquid recovery channel.

2. The microfluidic chip as described in claim 1, characterized in that, include: The mixing channel includes multiple mixing units, and the mixing units are connected to each other by a U-shaped channel; The mixing unit includes multiple mixing chambers connected in sequence. The shape of the mixing cavity is at least one of rhombus, Tesla valve shape and concentric circle.

3. The microfluidic chip as described in claim 2, characterized in that, The mixing and dilution chamber is provided with multiple first microcolumn structures; the cell fluid container chamber is provided with multiple second microcolumn structures; Multiple second micropillar structures are arranged in a one-to-one correspondence with multiple first micropillar structures, and the filter layer is located between multiple first micropillar structures and multiple second micropillar structures.

4. The microfluidic chip as described in claim 3, characterized in that, The liquid focusing segment includes: a first liquid focusing segment and a second liquid focusing segment; The first liquid focusing section includes multiple U-shaped bends connected end to end. The inlet of the first U-shaped curved flow channel is connected to the cell fluid accommodating cavity; The outlet of the last curved channel is connected to the inlet of the second liquid focusing section.

5. The microfluidic chip as described in claim 4, characterized in that, The second liquid focusing section is linear.

6. The microfluidic chip as described in claim 5, characterized in that, The mixing layer further includes a waste liquid outlet; the focusing layer further includes a drain hole; The input end of the waste liquid recovery channel is connected to the output end of the second liquid focusing section. After the output end converges, it is connected to the drain hole through the liquid outlet. The drain hole is connected to the waste liquid output channel, and the waste liquid output channel is connected to the waste liquid outlet.

7. The microfluidic chip as described in claim 1, characterized in that, The chip casing has a recessed mounting platform, and the filter layer is disposed within the mounting platform, located between the dilution layer and the focusing layer.

8. The microfluidic chip as described in claim 1, characterized in that, The chip casing is also provided with a viewing window, the position of which corresponds to the focusing channel of the focusing layer, for optical detection of cells.

9. The microfluidic chip as described in claim 1, characterized in that, The mixing layer, the dilution layer, the filter layer, and the focusing layer are connected by bonding.

10. The microfluidic chip as described in claim 1, characterized in that, It also includes guide grooves and alignment holes; The guide groove is disposed on the side wall of the chip housing and is used to cooperate with the guide structure of the cell counting device; The alignment wells sequentially penetrate the mixing layer, the dilution layer, and the focusing layer, and are used to align and fix the cell counting device.

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