Micro-fluidic chip

Abstract

The invention provides a micro-fluidic chip which comprises a uniform mixing layer, a chip dissolving cavity-dilution layer, a filtering layer and a focusing layer, cell sap and diluent can be preliminarily mixed by the uniform mixing layer and then fully diluted in the dilution layer, and the problem of non-uniformity possibly caused by single-time large-amplitude dilution is avoided. Then, the filter layer can effectively remove impurities and clotted cells. And finally, the focusing layer realizes cell focusing. The four functional layers of uniform mixing, diluting, filtering and focusing are sequentially stacked and integrated, so that the overall size of the microfluidic equipment is remarkably reduced, and high automation and optimization of the whole sample treatment process are realized.

Description

Technical Field

[0001] The present invention relates to the technical field of cell encapsulation, in particular to a microfluidic chip. Background Art

[0002] With the rapid development of biomedical technology, single-cell analysis technology plays an increasingly important role in disease diagnosis, drug development, and biological research. At present, commonly used cell analysis equipment mainly includes flow cytometers, one of whose core technologies is to achieve single-particle detection and precise detection of cells. Traditional flow cytometers use pressure-driven sheath flow focusing. Although it can achieve single-particle detection of cells, the equipment is bulky, expensive, and requires professional operation. In addition, existing technologies are difficult to adapt to cells of different sizes at the same time, which limits the scope of application of the equipment. Summary of the Invention

[0003] The present invention provides a microfluidic chip that integrates mixing, dilution, filtration and focusing functions, thereby achieving high integration of sample processing and reducing the volume of the microfluidic device.

[0004] The present invention provides a microfluidic chip, comprising:

[0005] A mixing layer is used to mix the cell solution and the diluent to obtain a cell mixture;

[0006] a dilution layer, used for diluting the cell mixture;

[0007] The filtering layer is used to filter the diluted cell mixture and transport the filtered cell mixture into the focusing layer;

[0008] A focusing layer, used to sort the cells in the cell mixture and arrange them in order to facilitate focusing and shooting of a cell counting device;

[0009] The dilution layer, the filter layer and the focusing layer are sequentially stacked on the mixing layer, and the mixing layer is arranged in the chip housing.

[0010] Furthermore, the mixing layer includes:

[0011] Cell solution input port, diluent input port and mixing flow channel;

[0012] The cell fluid input port and the diluent input port are connected to the inlets of the first channel and the second channel respectively;

[0013] The outlet of the first channel and the outlet of the second channel are both connected to the inlet of the mixing channel.

[0014] Furthermore, the mixing channel includes a plurality of mixing units, and any two of the mixing units are connected via a U-shaped channel;

[0015] The mixing unit comprises a plurality of mixing chambers connected in sequence.

[0016] Furthermore, the shape of the mixing chamber is at least one of a diamond shape, a Tesla valve shape and a concentric circle shape.

[0017] Furthermore, the dilution layer includes a liquid inlet and a mixing and dilution chamber, and a plurality of first micro-column structures are provided in the mixing and dilution chamber;

[0018] One end of the liquid inlet is connected to the outlet of the mixing channel;

[0019] The other end of the liquid inlet is communicated with the mixing and dilution chamber.

[0020] Furthermore, the focusing layer includes a liquid outlet and a cell fluid storage chamber, the liquid outlet is connected to the drainage hole of the dilution layer, a plurality of second micro-column structures are arranged in the cell fluid storage chamber, and the plurality of second micro-column structures are arranged in a one-to-one correspondence with the plurality of first micro-column structures, and the filtration layer is located between the plurality of first micro-column structures and the plurality of second micro-column structures.

[0021] Furthermore, the focusing layer further comprises a first liquid focusing segment and a second liquid focusing segment;

[0022] The first liquid focusing section includes a plurality of U-shaped curved flow channels connected end to end.

[0023] The inlet of the first U-shaped curved flow channel is connected to the cell fluid accommodating chamber;

[0024] The outlet of the last curved flow channel is connected to the inlet of the second liquid focusing section.

[0025] Furthermore, the second liquid focusing section is linear.

[0026] Furthermore, the mixing layer includes a waste liquid output channel and a waste liquid output port; the focusing layer also includes at least two waste liquid recovery channels;

[0027] The input ends of at least two of the waste liquid recovery channels are connected to the output end of the second liquid focusing segment, and the output ends are connected to the drainage hole through the liquid outlet after converging, the drainage hole is connected to the waste liquid output channel, and the waste liquid output channel is connected to the waste liquid output port.

[0028] Furthermore, the chip housing is provided with a sunken mounting platform, and the filter layer is provided in the mounting platform and is located between the dilution layer and the focusing layer.

[0029] Furthermore, a perspective window is provided on the chip housing, and the position of the perspective window corresponds to the focusing channel of the focusing layer, which is used for optical detection of cells.

[0030] Furthermore, the mixing layer, the dilution layer, the filtering layer and the focusing layer are connected by bonding.

[0031] Furthermore, it also includes a guide groove and an alignment hole;

[0032] The guide groove is provided on the side wall of the chip housing and is used to cooperate with the guide structure of the cell counting device;

[0033] The alignment holes sequentially penetrate the mixing layer, the dilution layer and the focusing layer, and are used to cooperate with the alignment and fixation of the cell counting device.

[0034] Compared with the prior art, the present invention has at least the following technical effects:

[0035] The microfluidic chip integration disclosed in the present invention includes a mixing layer, a dilution layer, a filtration layer and a focusing layer. Among them, the mixing layer can preliminarily mix the cell fluid and the diluent, and then fully dilute them in the dilution layer, avoiding the unevenness problem that may be caused by a single large dilution. Then, the filtration layer can effectively remove impurities and clumped cells. Finally, the focusing layer realizes cell focusing. By stacking and integrating the four functional layers of mixing, dilution, filtration and focusing in sequence, the overall volume of the microfluidic device is significantly reduced, and the automation and optimization of the entire sample processing process are realized. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is a schematic structural diagram of a microfluidic chip in one embodiment of the present invention;

[0037] Figure 2 is another schematic structural diagram of the microfluidic chip in one embodiment of the present invention;

[0038] Figure 3 is a schematic structural diagram of a mixing layer in one embodiment of the present invention;

[0039] Figure 4 is an enlarged view of a mixing channel in one embodiment of the present invention;

[0040] Figure 5 This is a schematic structural diagram of a dilution layer in one embodiment of the present invention;

[0041] Figure 6 is a schematic structural diagram of a focusing layer in one embodiment of the present invention;

[0042] Figure 7 An embodiment of the present invention is Figure 6 A magnified view of the structure in the middle.

[0043] Figure numerals: mixing layer 5, dilution layer 6, filtration layer 7, focusing layer 8, waste liquid output port 1, cell fluid input port 2, dilution liquid input port 3, outlet 55 of mixing channel, first channel 54, second channel 53, mixing channel 52, mixing chamber 522, U-shaped channel 521, mounting sink 62, liquid inlet 61, drainage hole 64, first microcolumn structure 65, mixing and dilution chamber 66, cell fluid accommodating chamber 81, second microcolumn structure 82, liquid outlet 86, first liquid focusing section 83, second liquid focusing section 84, waste liquid output channel 57, waste liquid recovery channel 85, perspective window 59, alignment hole 58, guide groove 500. DETAILED DESCRIPTION

[0044] The following description of a microfluidic chip of the present invention is provided in conjunction with a schematic diagram, which illustrates a preferred embodiment of the present invention. It should be understood that those skilled in the art may modify the present invention described herein while still achieving the advantageous effects of the present invention. Therefore, the following description should be understood as a general guideline for those skilled in the art and is not intended to limit the present invention.

[0045] The following paragraphs describe the present invention in more detail by way of example with reference to the accompanying drawings. The advantages and features of the present invention will become more apparent from the following description. It should be noted that the drawings are greatly simplified and not to exact scale, and are provided solely for the purpose of assisting in the description of the embodiments of the present invention.

[0046] Please refer to Figure 1-Figure 7 The present invention provides a microfluidic chip, comprising a mixing layer 5, a dilution layer 6, a filtration layer 7 and a focusing layer 8 stacked in sequence.

[0047] Specifically, the mixing layer 5 is used to mix the cell fluid and the diluent to obtain a cell mixture; the dilution layer 6 is used to dilute the cell mixture; the filtration layer 7 is used to filter the diluted cell mixture and transport the filtered cell mixture to the focusing layer 8; the focusing layer 8 is used to sort the cells in the cell mixture and arrange them in order to facilitate focusing and shooting of the cell counting device; wherein the dilution layer 6, the filtration layer 7 and the focusing layer 8 are stacked in sequence on the mixing layer 5, and the mixing layer 5 is arranged in the chip housing.

[0048] The microfluidic chip integration disclosed in the present invention includes a mixing layer 5, a dilution layer 6, a filtration layer 7 and a focusing layer 8, wherein the mixing layer 5 can preliminarily mix the cell fluid and the diluent, and then fully dilute them in the dilution layer 6, avoiding the unevenness problem that may be caused by a single large-scale dilution. Next, the filtration layer 7 can effectively remove impurities and clumped cells. Finally, the focusing layer 8 realizes cell focusing. By stacking and integrating the four functional layers of mixing, dilution, filtration and focusing in sequence, the overall volume of the microfluidic device is significantly reduced, and a high degree of automation and optimization of the entire sample processing process is achieved.

[0049] Please refer to Figure 3 In this embodiment, the mixing layer 5 further includes: a cell fluid input port 2 , a diluent input port 3 , a first channel 54 and a second channel 53 .

[0050] Specifically, the cell fluid input port 2 and the diluent input port 3 are respectively connected to the inlets of the first channel 54 and the second channel 53; the outlet of the first channel 54 and the outlet of the second channel 53 are both connected to the inlet of the mixing channel.

[0051] In the present embodiment, the dilution of the cell fluid is achieved by precisely controlling the flow ratio of the cell fluid and the diluent. Specifically, two independent peristaltic pumps or injection pumps are used to control the input of the cell fluid and the diluent, respectively. In a specific embodiment, the cell fluid is pumped in from the cell fluid input port 2, and the diluent is pumped in from the diluent input port 3, and the dilution multiple is controlled by adjusting the flow rate ratio of the two pumps. For example, when the cell fluid needs to be diluted 10 times, the flow rate of the diluent pump can be set to 9 times the flow rate of the cell fluid pump. After the two liquids are preliminarily mixed in the mixing channel, they enter the mixing and dilution chamber 66 for sufficient 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 multiples by simply adjusting the flow rate ratio of the two pumps according to the experimental requirements. The dilution multiple can be continuously adjusted within a wide range, and the dilution process is stable and controllable.

[0052] In one embodiment, the cell fluid inlet 2 and the diluent inlet 3 are each provided on a cylindrical boss structure. The top of the boss is provided with a through-hole for the introduction or removal of liquids, and the bottom of the boss is smoothly connected to the chip body. In addition, a rotary locking sleeve is provided on the top of the boss to achieve quick connection and sealing with external pipelines. It is understood that those skilled in the art can configure the cell fluid inlet 2 and the diluent inlet 3 with different structures according to actual conditions, and no specific limitations are imposed here.

[0053] In another specific embodiment, the cell fluid input port 2 and the diluent input port 3 are respectively arranged on one side edge of the mixing layer 5 shell, and a connection gap is provided between the two to ensure that the pipelines do not interfere with each other when connected.

[0054] Please refer to Figure 4 In this embodiment, the mixing channel 52 includes a plurality of mixing units, and each of the mixing units is connected via a U-shaped channel 521; the mixing unit includes a plurality of mixing chambers connected in sequence.

[0055] Specifically, after the cell fluid and diluent enter mixing channel 52, they first flow through multiple mixing chambers 522 within the first mixing unit. The continuous arrangement of mixing chambers 522 causes the fluid to undergo multiple divisions and reunions during flow, achieving initial mixing in a laminar state. Subsequently, the fluid enters the next mixing unit through U-shaped channel 521. The curved structure of U-shaped channel 521 induces secondary flow in the fluid, further disrupting the laminar flow interface.

[0056] In this embodiment, this solution combines the mixing unit with the U-shaped flow channel 521, and while retaining the advantage of low shear force, uses the geometric structure to actively induce fluid disturbance, thereby achieving efficient mixing in a shorter flow channel length and avoiding mechanical damage to cells caused by high-speed flow.

[0057] In another specific embodiment, the shape of the mixing unit is at least one of a diamond shape, a Tesla valve shape, and a concentric circle shape.

[0058] Specifically, the diamond-shaped mixing chamber, through its sudden expansion and contraction, generates a vortex effect, enhancing mixing effectiveness. The Tesla valve-shaped mixing chamber, with its valveless structure, generates directional vortices, improving mixing efficiency. Concentric circular mixing chambers create a rotating flow in the fluid, enhancing radial mixing. Those skilled in the art can select the mixing chamber structure based on their specific needs.

[0059] In another specific embodiment, the corners of the U-shaped flow channel 521 are designed with arc transitions to reduce resistance loss during the flow of liquid.

[0060] Please continue to refer to Figure 5 In this embodiment, the dilution layer 6 includes a liquid inlet 61 and a mixing and dilution chamber 66, and a plurality of first microcolumn structures 65 are provided in the mixing and dilution chamber 66; one end of the liquid inlet 61 is connected to the outlet 55 of the mixing channel; the other end of the liquid inlet 61 is connected to the mixing and dilution chamber 66.

[0061] In one embodiment, the plurality of first micro-columnar structures 65 are arranged in a lattice, with equal distances between any two of the first micro-columnar structures 65. This equidistant arrangement ensures uniform fluid resistance between the first micro-columnar structures 65, thereby ensuring substantially consistent flow rates and residence times within each first micro-columnar structure 65.

[0062] In another embodiment, the plurality of first micro-pillar structures 65 are arranged in a 7×3 lattice structure.

[0063] The focusing layer 8 includes a liquid outlet 86 and a cell fluid storage chamber 81. The liquid outlet 86 is connected to the drainage hole 64 of the dilution layer. A plurality of second microcolumn structures 82 are arranged in the cell fluid storage chamber 81. The plurality of second microcolumn structures 82 are arranged in a one-to-one correspondence with the plurality of first microcolumn structures 65. The filter layer 7 is located between the plurality of first microcolumn structures 65 and the plurality of second microcolumn structures 82.

[0064] Please refer to Figure 2 and Figure 5 In this embodiment, the chip housing is provided with a sunken mounting platform 62 , and the filter layer 7 is provided in the mounting platform 62 , between the dilution layer 6 and the focusing layer 8 .

[0065] Furthermore, in this embodiment, a porous membrane is provided in 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 advantages of selecting the porous membrane material of the above materials are that materials such as polycarbonate membrane, polyester membrane and silicon-based porous membrane have excellent chemical stability and mechanical strength, can maintain stable filtration performance under various working conditions, and have good biocompatibility.

[0066] It is understood that the pore size of the filter membrane is selected based on the size of the target cells, and the selection is based on intercepting large particulate matter and cell aggregates so that only single cells that meet the size requirements can pass through the filter layer and enter the subsequent focusing channel.

[0067] In a specific embodiment, for cells of 5 μm to 25 μm, the pore size of the filter membrane is generally set to 30 μm to 50 μm to effectively retain particles and cell aggregates larger than the target cell size, while allowing single target cells to pass through smoothly, thereby improving the normal operation of the subsequent focusing flow channel and the accuracy of cell counting.

[0068] Please refer to Figure 7Specifically, the first liquid focusing section 83 includes a plurality of U-shaped curved channels connected end to end. The inlet of the first U-shaped curved channel is connected to the cell fluid receiving chamber 81. The outlet of the last curved channel is connected to the inlet of the second liquid focusing section. The outlet of the last curved channel is connected to the inlet of the second liquid focusing section 84.

[0069] Furthermore, the focusing layer 8 also includes a first liquid focusing segment 83 and a second liquid focusing segment 84 .

[0070] In this embodiment, a plurality of U-shaped curved flow channels are provided in the first liquid focusing segment 83, and the liquid is driven to flow at high speed in the U-shaped curved flow channels by external power. Due to the inertial effect, a secondary flow is generated. This secondary flow generates a lateral force on the cells, causing the cells to gradually gather toward the liquid focusing segment. The first liquid focusing segment 83 is used to achieve the final focusing positioning of the cells at a stable flow rate, so that the cells move in a single row along the second liquid focusing segment 84. Compared with other focusing methods that require additional acoustic, electric or magnetic fields, this fluid dynamic focusing method can achieve cell focusing only by relying on the physical properties of the fluid itself, and has the characteristics of simple structure, convenient operation and easy control.

[0071] It is understood that the size and number of the U-shaped curved channels are configured based on the flow rate of the cell mixture and the size of the cells, and are not specifically limited here. Specifically, the parameters of the U-shaped curved channels are selected according to the following principles: the channel size is directly proportional to the target particle size, that is, the smaller the particle, the smaller the channel depth and width need to be; the number of channels is inversely proportional to the particle size, that is, the smaller the particle, the more U-shaped curved channels are required.

[0072] In a 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 flow channel is 30-40um, the width is 75-120um, and the number of the U-shaped curved flow channels is more than 5.

[0073] In another specific embodiment, when it is necessary to process smaller particles, such as platelets or certain large bacteria in the size range of 2μm to 10μm, the parameters of the focusing channel also need to be optimized accordingly. In order to generate effective inertial focusing force for these smaller particles, the channel size needs to be reduced proportionally. At this time, the depth of the U-shaped curved channel can be designed to be 15-25μm, and the width is correspondingly narrowed to 40-60μm. At the same time, in order to ensure that these tiny particles can obtain sufficient lateral migration in the fluid, the number of U-shaped curved channels usually needs to be increased, for example, increased to 8 or more, by accumulating more secondary flow effects, so as to achieve effective preliminary aggregation before entering the linear focusing section.

[0074] Furthermore, in this embodiment, the second liquid focusing section 84 is linear.

[0075] Specifically, the linear second liquid focusing section 84 has the advantage that, after the cells have initially gathered in the U-shaped curved channel at the front, they are already roughly pushed toward the center of the channel. The linear design avoids the complex secondary flows generated by the curve, thus stabilizing the flow rate. In this stable flow field, the initially gathered cells can ultimately be precisely positioned on the centerline of the channel, forming a single-file, evenly spaced queue.

[0076] Furthermore, in this embodiment, the mixing layer 5 includes a waste liquid output channel 57 and a waste liquid output port 1; the dilution layer 6 also includes a drainage hole 64; the focusing layer 8 also includes at least two waste liquid recovery channels 85;

[0077] The input ends of at least two of the waste liquid recovery channels 85 are connected to the output ends of the second liquid focusing section 84. After the output ends converge, they are connected to the drainage hole 64 through the liquid outlet 86. The drainage hole 64 is connected to the waste liquid output channel 57. The waste liquid output channel 57 is connected to the waste liquid output port 1.

[0078] Specifically, after focusing layer 8 completes cell inertial focusing, the unused mixed liquid enters the end of second liquid focusing section 84 and is then directed to waste liquid recovery channel 85. Waste liquid recovery channel 85 transports the liquid to drain hole 64, which vertically transfers the liquid to waste liquid output channel 57, ultimately draining the chip through waste liquid outlet 1. The entire waste liquid path adopts a hierarchical through-hole design to prevent liquid from being retained in a single plane.

[0079] In a specific embodiment, the number of the waste liquid recovery channels 85 can be 2, 3, or 5. After the multiple waste liquid recovery channels 85 converge, they are connected to the waste liquid output port 1 .

[0080] Specifically, the benefits of setting up multiple waste liquid recovery channels 85 are: first, multiple waste liquid recovery channels 85 can effectively disperse the waste liquid flow, avoid back pressure caused by excessive flow in a single channel, and thus improve the stability of liquid flow in the chip; second, when a waste liquid recovery channel 85 is blocked, other waste liquid recovery channels 85 can still work normally, thereby improving the reliability and robustness of the system; third, the parallel drainage method of multiple waste liquid recovery channels 85 can speed up the waste liquid discharge speed and improve the overall detection efficiency, which is particularly suitable for high-throughput cell detection applications; in addition, the convergence design of multiple channels also helps to balance the fluid resistance of each focusing channel, making the flow rate of each path in the multi-channel focusing system more uniform, thereby ensuring the consistency of the cell focusing effect.

[0081] In another specific embodiment, the waste liquid output channel 57 is W-shaped, and the waste liquid output channel 57 is connected to two waste liquid output ports 1.

[0082] In another specific embodiment, the waste liquid outlet 1 is a cylindrical boss structure, with a through hole at the top of the boss for the introduction or discharge of liquid, and the bottom of the boss smoothly transitions to the chip body. In addition, a rotary locking sleeve is provided at the top of the boss to achieve quick connection and sealing with external pipelines. It is understood that those skilled in the art can set the waste liquid outlet flow channel with different structures according to actual conditions, and no specific limitation is imposed here.

[0083] In another specific embodiment, the cell fluid input port 2 and the diluent input port 3 are respectively arranged on one side edge of the mixing layer 5 shell, and are respectively arranged on both sides of the cell fluid input port 2 and the diluent input port 3, and a connection gap is provided between the cell fluid input port 2 and the diluent input port 3 to ensure that the pipelines do not interfere with each other when connected.

[0084] In this embodiment, in order to balance the cell focusing effect and cell activity protection, the flow channel design needs to comprehensively consider the relationship between flow rate and shear force. Although the greater the flow rate, the better the focusing performance, the shear force that cells can tolerate is limited. Therefore, the center flow rate of the flow channel is generally kept below 1.2m / s to avoid damage to the cells. Under this flow rate limit, the system still needs to be able to achieve a higher detection flux. For example, when the center flow rate is 1.2m / s and for cells of 15μm in size, the theoretical maximum counting flux can reach 80,000 / s.

[0085] In order 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 filtration layer 7 and the focusing layer 8 are connected by bonding.

[0086] Through bonding connection, the overall pressure resistance of the chip can reach above 0.5Mpa, meeting the pressure requirements of high flow rate and high throughput detection.

[0087] In a specific embodiment, the bonding connection includes thermal compression bonding, ultrasonic bonding, etc., which are not specifically limited here. Those skilled in the art can select different bonding connection methods according to actual conditions, which are not specifically limited here.

[0088] Furthermore, in this embodiment, the chip housing is provided with a perspective window 59, which is positioned to correspond to the focused flow path of the focusing layer 8 and is used for optical detection of cells. When the chip is used in conjunction with a cell counter, the laser beam can pass through the perspective window 59 and illuminate the focused cell flow, enabling optical detection and counting of cells.

[0089] In a specific embodiment, the material of the perspective window 59 can be selected from optical glass, quartz glass, optical-grade PMMA, and other materials with good light transmittance, so as to accurately transmit the laser signal and improve the accuracy of cell detection.

[0090] Furthermore, in this embodiment, a guide groove 500 and an alignment hole 58 are included;

[0091] Specifically, the guide groove 500 is provided on the side wall of the mixing layer 5 for cooperating with the guide structure of the cell counting device.

[0092] The alignment holes 58 sequentially penetrate the mixing layer 5 , the dilution layer 6 , the filtration layer 7 and the focusing layer 8 , and are used for aligning and fixing the cell counting device.

[0093] In a specific embodiment, the guide groove 500 may be in the shape of a dovetail groove, a T-slot, an arc groove, or a stepped protrusion, and its size matches the corresponding guide structure on the cell counter.

[0094] In another specific embodiment, the cell counter can be stably connected to the alignment hole 58 by means of threaded connection, snap fixation, clamping or magnetic adsorption, so as to ensure accurate alignment and reliable fixation between the detection device and the chip.

[0095] In this embodiment, the cell counter is used in conjunction with the microfluidic chip, and precise alignment and fixation with the chip are achieved through the alignment holes 58 and guide grooves 500 on the chip. The cells passing through the focusing channel are counted, and the size, number and cell characteristics of the cells can be identified, thereby achieving accurate cell detection.

[0096] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A microfluidic chip, characterized in that: include: A mixing layer is used to mix the cell solution and the diluent to obtain a cell mixture; a dilution layer, used for diluting the cell mixture; The filtering layer is used to filter the diluted cell mixture and transport the filtered cell mixture into the focusing layer; A focusing layer, used to sort the cells in the cell mixture and arrange them in order to facilitate focusing and shooting of a cell counting device; The dilution layer, the filter layer and the focusing layer are sequentially stacked on the mixing layer, and the mixing layer is arranged in the chip housing.

2. The microfluidic chip according to claim 1, wherein The mixing layer comprises: Cell solution input port, diluent input port and mixing flow channel; The cell fluid input port and the diluent input port are connected to the inlets of the first channel and the second channel respectively; The outlet of the first channel and the outlet of the second channel are both connected to the inlet of the mixing channel.

3. The microfluidic chip according to claim 2, wherein: The mixing channel includes a plurality of mixing units, and each of the mixing units is connected by a U-shaped channel; The mixing unit comprises a plurality of mixing chambers connected in sequence.

4. The microfluidic chip according to claim 3, wherein The shape of the mixing chamber is at least one of a rhombus, a Tesla valve, and a concentric circle.

5. The microfluidic chip according to claim 2, 3 or 4, wherein: The dilution layer includes a liquid inlet and a mixing and dilution chamber, wherein a plurality of first micro-column structures are provided in the mixing and dilution chamber; One end of the liquid inlet is connected to the outlet of the mixing channel; The other end of the liquid inlet is communicated with the mixing and dilution chamber.

6. The microfluidic chip according to claim 5, characterized in that The focusing layer includes a liquid outlet and a cell fluid storage chamber. The liquid outlet is connected to the drainage hole of the dilution layer. A plurality of second microcolumn structures are arranged in the cell fluid storage chamber. The plurality of second microcolumn structures are arranged in a one-to-one correspondence with the plurality of first microcolumn structures. The filtration layer is located between the plurality of first microcolumn structures and the plurality of second microcolumn structures.

7. The microfluidic chip according to claim 6, wherein: The focusing layer further includes a first liquid focusing segment and a second liquid focusing segment; The first liquid focusing section includes a plurality of U-shaped curved flow channels connected end to end. The inlet of the first U-shaped curved flow channel is connected to the cell fluid accommodating chamber; The outlet of the last curved flow channel is connected to the inlet of the second liquid focusing section.

8. The microfluidic chip according to claim 7, wherein: The second liquid focusing section is linear.

9. The microfluidic chip according to claim 8, wherein The mixing layer includes a waste liquid output channel and a waste liquid output port; the focusing layer also includes at least two waste liquid recovery channels; The input ends of at least two of the waste liquid recovery channels are connected to the output end of the second liquid focusing segment, and the output ends are connected to the drainage hole through the liquid outlet after converging, the drainage hole is connected to the waste liquid output channel, and the waste liquid output channel is connected to the waste liquid output port.

10. The microfluidic chip according to claim 1, wherein: The chip housing is provided with a sunken installation platform, and the filter layer is arranged in the installation platform and is located between the dilution layer and the focusing layer.

11. The microfluidic chip according to claim 1, wherein The chip housing is further provided with a perspective window, the position of which corresponds to the focusing channel of the focusing layer and is used for optical detection of cells.

12. The microfluidic chip according to claim 1, wherein: The mixing layer, the dilution layer, the filtering layer and the focusing layer are connected by bonding.

13. The microfluidic chip according to claim 1, wherein: It also includes guide grooves and alignment holes; The guide groove is provided on the side wall of the chip housing and is used to cooperate with the guide structure of the cell counting device; The alignment holes sequentially penetrate the mixing layer, the dilution layer and the focusing layer, and are used to cooperate with the alignment and fixation of the cell counting device.

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