A microfluidic chip for cell counting and typing and its sampling method

Abstract

The present invention provides a microfluidic chip for cell counting and typing and a sampling method thereof, belonging to the field of biomedical detection technology. The sampling and detection chamber of the microfluidic chip comprises a sampling port, one or more detection areas, and a flow guide groove area connecting the sampling port and the detection area. The thickness of the detection area is smaller than that of the flow guide groove area, and a plurality of exhaust holes and a sampling notch are provided on the detection area or the flow guide groove area. The chip can realize two sampling modes, active aspiration and passive injection, thereby avoiding the generation of bubbles, achieving precise control of the cell sample amount, and taking into account the precision of cell counting and typing detection at the same time, laying a foundation for on-site cell analysis in biomedical research and clinical testing applications.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical detection, and particularly relates to a microfluidic chip for cell counting and typing and a sampling method thereof. Background Art

[0002] In biomedical research and clinical testing applications, cells need to be analyzed to obtain relevant information for basic research or physical status. Commonly used instruments are flow cytometers and fully automatic hematology analyzers. In terms of function, they are mainly based on cell count measurement (i.e., "counting") and type differentiation (i.e., "typing").

[0003] Among them, the core principle of the flow cytometer is to use the sample tube and sheath fluid tube of the liquid flow system to make the sheath fluid surround the sample to be tested and flow at high speed in a circular stream, so as to arrange the cells to be tested in a single row and pass through the laser irradiation area in sequence. By analyzing the forward scattered light, side scattered light, and side fluorescence of each cell one by one, the number and type information of the cells can be finally obtained.

[0004] The mainstream counting principle of fully automated hematology analyzers is the Coulter principle (also known as the electrical impedance method). This principle states that when cells suspended in an electrolyte pass through a small orifice tube with the electrolyte, they displace the same volume of electrolyte. In a constant current circuit, this causes a transient change in the resistance between the two electrodes inside and outside the orifice tube, generating potential pulses whose magnitude and frequency are proportional to the size and number of cells, thereby achieving cell counting. Before the Coulter method, selective hemolytic agents or chemical reagents are used to disrupt other cells while leaving specific cells intact. This, combined with the counting function of the Coulter method, allows for typing and counting.

[0005] Both methods analyze single cells and can therefore achieve accurate cell counting and typing. However, both methods suffer from the problems of expensive instruments, large size, and specialized operation, making them unsuitable for situations where cell analysis is required on-site or in non-specialized settings. For example, in specific biomedical research, there are situations where the conditions for bringing cell samples to be tested back to the laboratory are not available and immediate analysis is required on-site, or where communities, families, village doctors, etc. need to perform blood cell analysis in other settings other than laboratory departments or central laboratories. These methods are not conducive to promotion and application.

[0006] In view of the above problems, in clinical testing applications, existing testing equipment uses disposable counting pieces (see Figure 1), the core structure of the counting sheet is an open measuring cavity 01 formed by two parallel transparent sheets at a certain distance and surrounded by a bottom, and the measuring cavity has a measuring area 02 with a thickness further reduced to about 100 μm. The measuring cavity has the following functions: (1) Reagent fixation: red blood cell hemolysis agents (quaternary ammonium salts, saponins, etc.) and white blood cell color dyes (hematoxylin, methylene blue, etc.) are fixed on the inner wall of the measuring cavity; (2) Blood processing: The blood sample enters the measuring cavity by active suction, and red blood cell hemolysis and white blood cell staining are completed in the measuring cavity; (3) Cell spreading: The thickness of the detection area is further reduced, so that the white blood cells in the sample are spread as much as possible in a single layer in the measuring area, which is convenient for single cell analysis; (4) Detection connection: The detection system can obtain the image of the single-layer spread cells in the measuring area through the side wall of the measuring cavity. Therefore, the counting of cells and the typing based on the recognition of cell morphology are achieved by relying on color dyes. However, the counting sheet has the following defects in actual use:

[0007] (1) The use of a single-thickness detection area makes it impossible to simultaneously meet the precision requirements of cell counting and typing: The counting slide has a single-thickness measurement area, which makes it impossible to simultaneously meet the requirements of a large number of cells per unit area in counting and the high precision requirements of individual cells per unit area in typing, thus affecting the precision of counting and typing.

[0008] (2) Bubbles are easily generated during sample injection, affecting the accuracy of subsequent measurements: the structure of the counting plate does not take into account the design to avoid the generation of bubbles. The presence of bubbles will seriously interfere with the subsequent accurate analysis;

[0009] (3) After injection, the sample is prone to wiping loss and movement outflow loss, making it impossible to guarantee the accuracy of the sample amount.

[0010] (4) Limited sampling methods and application scenarios: The structure of the counting plate is not compatible with both active sampling and passive sampling. The sample and reagent can only be mixed in the measuring chamber, which limits the scope of application. Summary of the Invention

[0011] In order to solve the above problems, the present invention provides an improved microfluidic chip for cell counting and typing.

[0012] The technical solution adopted in the present invention is:

[0013] A microfluidic chip for cell counting and typing, comprising a supporting substrate (1) and a sampling and detection cavity (2) provided on the supporting substrate (1); the sampling and detection cavity (2) is a semi-open cavity formed by two parallel chip side walls (4) with a certain gap, and comprises a detection area (5), a sampling port (7), and a guide groove area (6) connecting the sampling port (7) and the detection area (5); the thickness H of the detection area (5) is 1 / 4 of the upper and lower portions thereof. 检Less than the thickness H of the guide groove area (6) 导 The sampling and detection chamber (2) is provided with at least one exhaust hole (8), which is a through hole connecting the interior of the sampling and detection chamber (2) with the outside atmosphere, and which passes through one side wall of the detection area (5) or the guide groove area (6) or symmetrically passes through both side walls of the detection area (5) or the guide groove area (6).

[0014] In the above-mentioned microfluidic chip for cell counting and typing, the exhaust hole (8) is an inverted conical through hole, the small end of the cone opens toward the inside of the detection area (5) or the guide groove area (6), and the large end of the cone opens toward the outside atmosphere.

[0015] In the above-mentioned microfluidic chip for cell counting and typing, a detection area (5) is provided in the sampling and detection cavity (2), and the detection area (5) has a single thickness H 检 .

[0016] In the above-mentioned microfluidic chip for cell counting and typing, the sampling and detection cavity (2) is provided with a plurality of detection areas (5), the plurality of detection areas (5) are independent and connected to each other, and have the same or different thicknesses, and the thickness of any detection area (5) is less than the thickness H of the guide groove area (6). 导 .

[0017] In the above-mentioned microfluidic chip for cell counting and typing, the sampling detection cavity (2) is provided with two detection areas (5) of different thicknesses, namely a first detection area (51) and a second detection area (52), and the first detection area (51) and the second detection area (52) are connected by a guide groove area (6).

[0018] In the above-mentioned microfluidic chip for cell counting and typing, the sampling port (7) is located at the opening of the upper edge of the two chip side walls of the sampling detection cavity (2), and a sampling notch (9) is provided at the upper edge of one of the two chip side walls (4) located at the sampling port (7) to inject a liquid sample through the sampling notch (9).

[0019] In the above-mentioned microfluidic chip for cell counting and typing, the sampling port (7) is in a concave arc shape, and the angle α between the tangent of the downward arc and its horizontal reference plane is in the range of 15° to 45°.

[0020] In the above-mentioned microfluidic chip for cell counting and typing, each detection area is provided with a corresponding sampling port.

[0021] In the above-mentioned microfluidic chip for cell counting and typing, each sampling port is provided with a sampling notch.

[0022] In the above-mentioned microfluidic chip for cell counting and typing, the inner edge of the end of the chip side wall (4) is provided with a transition fillet (10), and the range of the transition fillet R is 0.2mm-1.5mm.

[0023] The microfluidic chip for cell counting and typing further comprises a sample processing chamber (3) arranged on the supporting substrate (1), which serves as a storage container for the reagent and a mixing operation container for the reagent and the liquid sample to be tested.

[0024] In the above-mentioned microfluidic chip for cell counting and typing, the supporting substrate (1) is the handheld part of the microfluidic chip, and the sampling and detection cavity (2) is arranged at the front end of the supporting substrate (1) and is integrally formed with the supporting substrate (1) or bonded to the supporting substrate (1).

[0025] In the above-mentioned microfluidic chip for cell counting and typing, the detection area (5) is a combination of a rectangle, a square, a trapezoid, a circle or an arc and other shapes, and each shape can be provided with rounded corners, right angles or a combination of rounded corners and right angles.

[0026] The microfluidic chip for cell counting and typing is used in conjunction with a detection and analysis instrument for cell counting and typing. The support substrate (1) is further provided with a positioning groove, which is arranged below the support substrate (1) and is used for alignment and positioning with the detection and analysis instrument.

[0027] The above-mentioned microfluidic chip for cell counting and typing also includes a pre-packaged liquid reagent, which is pre-packaged in the following manner: the liquid reagent is added to the sampling detection cavity of the microfluidic chip through the sampling port or sampling notch of the microfluidic chip, dried or freeze-dried, and the reagent is pre-packaged in the detection cavity; or, the liquid reagent is added to the sample processing cavity of the microfluidic chip, dried or freeze-dried, and the reagent is pre-packaged in the sample processing cavity.

[0028] The present invention also provides a sampling method using the above-mentioned microfluidic chip for cell counting and typing, comprising:

[0029] The sampling port of the microfluidic chip is immersed in a liquid sample for sampling, so that the liquid sample flows from the guide groove area (6) into the detection area (5) under the action of capillary force and fills the detection area (5), wherein the capillary force and the thickness H of the detection area (5) are 检 , the thickness H of the guide groove area (6) 导 The following relationship is satisfied:

[0030]

[0031] Another sampling method using the above-mentioned microfluidic chip for cell counting and typing comprises:

[0032] Immerse the sampling port in the liquid sample for sampling, so that the liquid sample flows from the guide groove area into the detection area under the action of capillary force and fills it; or, inject the liquid sample into the sampling detection cavity through the sampling notch for sampling, so that the liquid sample flows from the guide groove area into the detection area and fills it.

[0033] The beneficial effects of the present invention are as follows: the microfluidic chip of the present invention is provided with a semi-open sampling and detection cavity, the sampling and detection cavity has a sampling port, a detection area, and a guide groove area connecting the sampling port and the detection area, the thickness of the detection area is less than the thickness of the guide groove area, and multiple exhaust holes are provided on the guide groove area and / or the detection area. By providing the sampling notch, the exhaust holes, and one or multiple detection areas with different thicknesses, the microfluidic chip can realize two sampling methods, active suction and passive injection, thereby avoiding the generation of bubbles, achieving precise control of the cell sample amount, and taking into account the precision of cell counting and typing detection at the same time, laying the foundation for on-site cell analysis in biomedical research and clinical testing applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a schematic diagram of the structure of an existing disposable counting sheet for white blood cell typing;

[0035] Figure 2A 1 is a schematic diagram of the planar structure of the microfluidic chip according to the first embodiment of the present invention;

[0036] Figure 2B Schematic diagram of the three-dimensional structure of the embodiment 1 of the microfluidic chip of the present invention

[0037] Figure 2C yes Figure 2A The cross-sectional view taken along line AA;

[0038] Figure 3A 1 is a schematic diagram of the planar structure of the second embodiment of the microfluidic chip of the present invention;

[0039] Figure 3B 1 is a schematic diagram of the three-dimensional structure of the microfluidic chip according to the second embodiment of the present invention;

[0040] Figure 3C yes Figure 3A A cross-sectional view taken along line BB;

[0041] Figure 4A 1 is a schematic diagram of the planar structure of the microfluidic chip according to the third embodiment of the present invention;

[0042] Figure 4B 1 is a schematic diagram of the three-dimensional structure of the microfluidic chip of embodiment 3 of the present invention;

[0043] Figure 4C yes Figure 4A A cross-sectional view taken along line C1-C1;

[0044] Figure 4D yes Figure 4A A cross-sectional view taken along line C2-C2;

[0045] Figure 5A 1 is a schematic diagram of the planar structure of a fourth embodiment of the microfluidic chip of the present invention;

[0046] Figure 5B 1 is a schematic diagram of the three-dimensional structure of the fourth embodiment of the microfluidic chip of the present invention;

[0047] Figure 5C It is a structural schematic diagram of the sampling and detection cavity in the fourth embodiment of the microfluidic chip of the present invention.

[0048] The reference numerals in the figures are as follows:

[0049] 01- measurement cavity, 02- measurement area;

[0050] 1-support substrate; 2-sampling detection cavity;

[0051] 5-detection area, 51-first detection area, 52-second detection area; 6-guiding groove area;

[0052] 7-sampling port, 71-first sampling port, 72-second sampling port; 9-sampling notch, 91-first sampling notch, 92-second sampling notch; 8-exhaust hole, 81-first exhaust hole, 82-second exhaust hole, 83-third exhaust hole, 84-fourth exhaust hole;

[0053] 3-sample processing chamber; 4-chip side wall, 10-transition fillet, 11-liquid bridge surface. DETAILED DESCRIPTION

[0054] To address the problems of existing disposable white blood cell counting slides, such as the inability to simultaneously achieve both counting and typing precision, bubble interference, low sampling accuracy, a single sampling method, and a limited scope of application, the present invention provides a microfluidic chip for cell counting and typing and a sampling method thereof. The microfluidic chip comprises a supporting substrate and a sampling and detection cavity fixed to the supporting substrate. The sampling and detection cavity is a semi-open cavity formed by two parallel chip sidewalls with a certain gap. The chip features multi-mode sampling, a design of multiple detection areas of one or different thicknesses, a guide channel area for connecting the sampling port and the detection area, and an exhaust hole design. By providing the sampling notch, the exhaust hole, and the design of one or multiple detection areas of different thicknesses, the microfluidic chip can implement both active aspiration and passive injection sampling modes, avoid the generation of bubbles, achieve precise control of cell sample volume, and simultaneously achieve both precision in cell counting and typing detection, laying a foundation for on-site cell analysis in biomedical research and clinical testing applications.

[0055] The microfluidic chip for cell counting and typing and the sampling method thereof of the present invention are described in detail below with reference to Examples 1 to 4 and the accompanying drawings.

[0056] Example 1

[0057] Figure 2A-2CThis is an example of the structure of the microfluidic chip of the present invention. Figure 2A-2C In the illustrated embodiment 1, the microfluidic chip includes a supporting substrate 1 and a sampling detection cavity 2 disposed on the supporting substrate 1, wherein the supporting substrate 1 is the handheld portion of the microfluidic chip, which is designed to be suitable for handholding. For example, in the embodiment 1, the handheld portion is rectangular, and a measuring cavity with an arc-shaped edge extends from its front end. In the present invention, the measuring cavity is the sampling detection cavity 2; the sampling detection cavity 2 can be integrally formed with the supporting substrate 1, or the sampling detection cavity 2 is bonded to the front end of the supporting substrate 1. The sampling detection cavity 2 is a semi-open cavity formed by two parallel chip side walls 4 with a certain gap, including a sampling port 7, a sampling notch 9, a detection area 5, and a guide groove area 6 connecting the sampling port 7 and the detection area 5, wherein:

[0058] The sampling port 7 is located at the upper edge opening of the two chip side walls 4 of the sampling detection cavity 2. Figure 1 Similar to the existing counting slice shown, samples can be added by active suction through the sampling port 7.

[0059] The detection area 5 is located in the sampling detection cavity 2. The shape of the detection area 5 can be a rectangle, a square, a trapezoid, a circle, or a combination of arcs and other shapes. Each shape can have rounded corners, right angles, or a combination of rounded corners and right angles. The present invention does not limit the specific shape of the detection area 5. The detection area 5 has a single thickness H 检 The sample enters the detection area 5 to form a detection surface. The thickness of the detection area 5 is generally in the range of 60μm-120μm. The detection area 5 with a large thickness has a large sample carrying capacity per unit area of ​​the detection surface and a large depth of field, which is suitable for the overall accurate measurement of the number of cells; the detection area 5 with a small thickness has a large spreading area of ​​the same volume of liquid sample on the detection surface, which is suitable for the precise distinction of cell types.

[0060] The guide groove area 6 is located in the sampling and detection cavity 2, and is connected to the sampling port 7 and the detection area 5. The thickness of the guide groove area 6 is generally in the range of 120μm to 500μm. Figure 2C As shown, the thickness of the detection area 5 is smaller than that of the guide groove area 6 , and the liquid sample enters the flow path formed by the guide groove area 6 from the sampling port 7 and is evenly and quickly introduced into the detection area 5 and fills it.

[0061] The thickness of the detection zone 5 and the thickness of the flow channel 6 determine the flow and spread of the liquid sample in the flow channel 6. The liquid sample enters the flow channel 6 through the sampling port 7. The sample is drawn into the flow channel 6 during a purely inertial rise phase under the action of capillary force. The relationship between the volume of the drawn liquid sample and the thickness of the detection zone 5 can be obtained using Equation 1 for the purely inertial rise phase of capillary flow:

[0062]

[0063] In order to ensure that the liquid sample flows continuously from the guide groove area 6 into the detection area 5 and fills it under the action of capillary force, the capillary force is required to be greater than zero. 检 , the thickness of the guide groove area H 导 The following relationship exists:

[0064]

[0065] Specifically, in this embodiment, the detection area 5 is provided with one, which is located in the sampling detection cavity 2 and has a single thickness H 检 , its shape is a rounded rectangle. When the thickness H of the detection area 5 检 When the thickness is large, the detection surface formed on the chip side wall 4 has a large depth of field and a large sample carrying capacity per unit area, which is suitable for the overall accurate measurement of the number of cells. The thickness of the detection area 5 is preferably 90μm-120μm; when the thickness H of the detection area 5 is 检 When the thickness is small, the depth of field of the detection surface formed on the chip sidewall 4 is small and the sample spreading area per unit volume is large, which is suitable for precise differentiation of cell types. The thickness of the detection area 5 is preferably 60 μm-90 μm.

[0066] Furthermore, a sampling notch 9 is provided on the upper edge of one of the two chip side walls 4 at the sampling port 7, which facilitates the addition of samples through the sampling notch 9 by injection. The sampling port 7 and the sampling notch 9 are compatible with both active injection and passive aspiration liquid sampling modes. In this embodiment, the sampling port 7 is concavely arc-shaped, and the angle α between the tangent of its downward arc (the left arc in the figure) and the horizontal reference plane of the sampling port 7 is (see Figure 2A ), can determine the direction of the flow of the liquid sample to be tested into the guide groove area 6, so as to ensure that the liquid sample to be tested spontaneously flows into the detection area 5 in a predetermined manner and fills the detection area 5. The preferred range of angle α is 15° to 45°.

[0067] Specifically, the guide groove area 6 is located in the sampling and detection cavity 2, connecting the sampling port 7 and the detection area 5, and the thickness H of the guide groove area 6 is 导 Greater than the thickness H of the detection area 5 检 , try to avoid bubbles during the injection process; to ensure that the liquid sample to be tested can continuously flow from the guide groove area 6 into the detection area 5 under the action of capillary force and fill it, the capillary pressure is required to be greater than zero. The calculation of the capillary force refers to formula 2).

[0068] The structural design of the first embodiment is suitable for cell counting and typing detection with a small number of cells per unit volume or low cell abundance, or for applications where only cell counting or cell typing is required. According to the application, a detection area 5 with a single thickness is selected and an appropriate thickness H is set. 检 , in order to perform high-precision measurement of cell number or high-precision analysis of single cell types.

[0069] Of course, in this embodiment, one detection area 5 may also have multiple thicknesses H 检1 , H 检2 The capillary force that drives the liquid sample to be tested into the sampling and detection cavity 2 is proportional to the thickness of the detection area 5 and the thickness of the guide groove area H. 导 The relationship still satisfies Equation 2). When the sample enters the detection area 5 with varying thickness, detection surfaces with different depths of field and different spreading states can be formed. Data processing of cells in detection areas of varying thickness can simultaneously take into account the precision measurement of cell counting and typing.

[0070] Example 2

[0071] Figure 3A-3C The structure of the microfluidic chip of the present invention is shown in the second embodiment. The structure of the second embodiment is a further improvement on the structure of the first embodiment, and the difference between the second embodiment and the first embodiment is that:

[0072] In order to further avoid the generation of bubbles, at least one exhaust hole 8 is provided on the sampling detection chamber 2. The exhaust hole 8 is a through hole connecting the interior of the sampling detection chamber 2 with the outside atmosphere. The exhaust hole 8 can be located on one side or both sides of the detection area 5 or the guide groove area 6. It can be a symmetrical or asymmetrical through hole, that is, the exhaust hole 8 passes through the side wall of the detection area (5) or the guide groove area (6) or symmetrically passes through the two side walls of the detection area (5) or the guide groove area (6). Preferably, the exhaust hole 8 is an inverted cone-shaped through hole, that is, the small end of the cone opens toward the inside of the guide groove area 6, and the large end of the cone opens toward the outside atmosphere (see Figure 3C ).

[0073] The advantages of the inverted cone exhaust hole are that, first, it utilizes the surface tension between the liquid sample to be tested and the gas to make it easier to discharge bubbles. The exhaust method based on the inverted cone hole proposed in the present invention is not affected by the sampling angle and distance, and can effectively discharge bubbles under different sampling methods; when the reagent needs to be pre-packaged in the sampling detection chamber 2, since the contact area between the reagent and the external environment is very small, when the reagent is added to the sampling detection chamber 2, its drying process is long. The design of the exhaust hole 8 can increase the contact area between the reagent and the external environment, accelerate the drying and uniform distribution of the reagent, thereby avoiding the generation of bubbles in many cases; for the purpose of accurately controlling the amount of sample, after the liquid sample to be tested enters the exhaust hole, it is not easy to overflow due to the effect of surface tension; the exhaust hole 8 is set on one side or both sides of the sampling detection chamber 2. After the sampling is completed, only the side of the chip needs to be wiped to avoid the loss of liquid sample due to wiping the sampling port; at the same time, the inverted cone hole structure further reduces the possibility of liquid wiping loss.

[0074] The chip's sidewalls 4 can be used for subsequent cell counting and typing monitoring and analysis. To prevent the liquid sample from escaping due to gravity when the microfluidic chip is moved, the inner edges of the chip's sidewalls 4 are provided with a transition radius 10. The transition radius R ranges from 0.2mm to 1.5mm. When the microfluidic chip is loaded, the liquid sample forms a stable liquid bridge 11 at the transition radius 10 on the two chip sidewalls 4, effectively balancing the gravity of the liquid sample and preventing it from escaping.

[0075] Specifically, such as Figure 3C As shown, there are two exhaust holes 8, namely the first exhaust hole 81 and the second exhaust hole 82, which are respectively located at the front end (the position before the liquid sample to be tested enters the detection area 5) and the rear end (the position after the liquid sample to be tested flows out of the detection area 5) of the guide groove area 6, and are symmetrical inverted cone-shaped through holes that pass through the guide groove area 6, which can further avoid the generation of bubbles.

[0076] The other structures of the second embodiment are the same as those of the first embodiment. For technical solutions not mentioned in the second embodiment, please refer to the first embodiment and will not be repeated here.

[0077] Example 3

[0078] Figures 4A-4D The structure of the microfluidic chip of the present invention is shown in the following figure. The structure of the third embodiment is a further improvement on the structure of the second embodiment and / or the first embodiment. The difference between the third embodiment and the second embodiment and / or the first embodiment is that:

[0079] In this embodiment, there are two detection areas 5, namely the first detection area 51 and the second detection area 52, and the two detection areas are independent of each other (i.e., arranged at a distance) and connected, and the two detection areas are connected by the guide groove area 6. In this embodiment, the two detection areas are both rectangular with a combination of rounded corners and right angles, with thicknesses of H and H. 检1 、H 检2 , wherein the thickness H of the first detection area 51 is 检1 The depth of field of the detection surface formed by the liquid sample to be tested in the first detection area 51 is large, and the sample carrying capacity per unit area is large, which is suitable for the overall accurate measurement of the number of cells; the thickness H of the second detection area 52 is large. 检2 The depth of field of the detection surface formed by the liquid sample to be tested in the second detection area 52 is small, and the sample spreading area per unit volume is large, which can be used for precise differentiation of cell types. This embodiment sets two detection areas 51 and 52 of different thicknesses to simultaneously take into account the precision measurement of cell counting and typing. The two detection areas 51 and 52 are connected by a guide groove area 6 of uniform thickness. Of course, the thickness H of the two detection areas is 检1 、H 检2 are smaller than the thickness H of the guide groove area 导 .

[0080] Specifically, in this embodiment, there are four exhaust holes 8, namely the first exhaust hole 81, the second exhaust hole 82, the third exhaust hole 83, and the fourth exhaust hole 84, wherein the first exhaust hole 81 and the second exhaust hole 82 are respectively located at the front end (the position before the liquid sample to be tested enters the detection area 51) and the rear end (the position after the liquid sample to be tested flows out of the detection area 52) of the guide groove area 6, the third exhaust hole 83 is arranged on the first detection area 51, and the fourth exhaust hole 84 is arranged on the second detection area 52. The exhaust holes 8 are all symmetrical inverted cone-shaped through holes that pass through the guide groove area 6 or the detection area 5, which can further avoid the generation of bubbles.

[0081] Obviously, multiple detection areas 5 can also have the same thickness, which is smaller than the thickness of the guide groove area 6. By setting different detection areas 5, it is helpful to analyze the accuracy and consistency of the detection results of each detection area 5.

[0082] The other structures of the third embodiment are the same as those of the second embodiment and / or the first embodiment, and are not described again here.

[0083] Example 4

[0084] Figures 5A-5C The structure of the fourth embodiment of the microfluidic chip of the present invention is shown. The structure of the fourth embodiment is a further improvement on the structure of the third embodiment, and its difference from the third embodiment is that:

[0085] In this embodiment, there are two sampling ports 7, namely the first sampling port 71 and the second sampling port 72. A first sampling notch 91 is provided on one side wall of the corresponding first sampling port 71, and a second sampling notch 92 is provided on one side wall of the second sampling port 72. A sampling port (the first sampling port 71 and the second sampling port 72) is provided for each detection area (the first detection area 51 and the second detection area 52). For a detection area with a larger thickness, a larger liquid sample carrying capacity per unit area is required. By adding corresponding sampling ports, the insufficient amount of liquid sample caused by a single sampling port can be avoided.

[0086] Specifically, multiple guide groove areas 6 can be set (guide groove areas of different thicknesses are called different guide groove areas, and the guide groove areas 6 are interconnected), each guide groove area 6 corresponds to the corresponding detection area 5, so as to facilitate the controlled and rapid flow of the liquid sample into the corresponding detection area 5. By setting guide groove areas 6 and detection areas 5 of different thicknesses, different liquid flow rates and liquid laminar flow characteristics can be adjusted, and the thickness of the detection area 5 is less than the thickness of all guide groove areas 6; similarly, the guide groove area 6 can be set to one (guide groove areas 6 of the same thickness and connected are called the same guide groove area), and multiple detection areas 5 correspond to the same guide groove area 6.

[0087] Specifically, by adjusting the angle α between the tangent line of the downward arc of the sampling port 7 and the horizontal reference plane of the sampling port 7, the specific direction at which the liquid sample to be tested enters the flow channel area 6 can be specified. This ensures that the liquid sample to be tested flows spontaneously into the detection area 5 in a specific manner until it is full. The preferred range of angle α is 15° to 45°.

[0088] Based on the above embodiments, the microfluidic chip of the present invention further includes the following designs:

[0089] A sample processing chamber 3 is provided on the support substrate 1 of the microfluidic chip. The sample processing chamber 3 can have any shape (e.g., cylindrical, inverted truncated cone, etc.) and can serve as a storage container for dry or liquid reagents and a mixing container for reagents and liquid samples to be tested, simplifying on-site testing. Specifically, the handheld end of the support substrate 1 has a certain thickness (which is greater than the thickness of the sampling and detection chamber 2). The support substrate 1 is provided with an inverted truncated cone-shaped groove as the sample processing chamber 3, which is used for pretreatment of the cell sample to be tested.

[0090] A positioning groove is provided on the microfluidic chip's supporting substrate 1 to facilitate its use with a cell counting and typing detection and analysis instrument. This groove is located below the microfluidic chip's supporting substrate 1, preferably below the sample processing chamber 3 at the rear end of the supporting substrate 1, for alignment and positioning with the detection and analysis instrument. When the microfluidic chip is placed in the detection and analysis instrument, the positioning groove mates with a protrusion on the supporting platform within the instrument, thereby achieving alignment and positioning.

[0091] The microfluidic chip material of the present invention can be selected from any one or more combinations of optical-grade transparent polymers, glass, and quartz to ensure high light transmittance and low spontaneous fluorescence performance.

[0092] Obviously, the structure of the microfluidic chip of the present invention is not limited to the structure described in the above embodiments. On the basis of the concept of the present invention, simply increasing or decreasing the number of detection areas, guide groove areas, sampling ports (with sampling notches), and exhaust holes, changing the shape, position or combination form, all fall within the disclosure of the present invention.

[0093] The method for using the above-mentioned microfluidic chip is as follows:

[0094] Pre-packaging of liquid reagents; specifically, there are two ways to pre-packaging liquid reagents:

[0095] First, the liquid reagent to be pre-packaged is added to the sampling and detection chamber 2 of the microfluidic chip through the sampling port 7 or sampling notch 9 of the microfluidic chip and then dried or freeze-dried. The reagent is pre-packaged in the detection chamber. When testing is required, the liquid sample is drawn into or injected into the sampling and detection chamber 2 through the sampling port 7 or sampling notch 9. This means that the liquid sample is added and the reaction between the sample and the reagent is completed simultaneously in the detection chamber.

[0096] Second, the liquid reagent to be pre-packaged is added to the sample processing chamber 3 of the microfluidic chip and dried or freeze-dried. The reagent is pre-packaged in the sample processing chamber. When testing, the liquid sample is added to the sample processing chamber 3. The sample and reagent react, and the resulting mixed solution is then added to the sampling detection chamber 2 of the microfluidic chip through the sampling port 7 or sampling notch 9.

[0097] There are two ways to add samples to the microfluidic chip: one is active sampling based on capillary force, that is, immersing the sampling port 7 of the microfluidic chip into the liquid sample or reagent, and actively sucking the liquid through capillary force until it fills the detection area 5; the other is passive sampling based on a pipette, that is, using a pipette to suck the original liquid sample or reagent, placing the pipette at the sampling notch 9 of the sampling port 7 of the microfluidic chip, and adding the liquid to the detection area 5 of the microfluidic chip until it is full.

[0098] In summary, the present invention has the following outstanding features and effects:

[0099] 1) The sampling and detection cavity 2 of the present invention adopts a semi-open cavity structure. When reagent pre-packaging is required, the unobstructed air path accelerates the speed and uniformity of reagent packaging, preventing the formation of bubbles due to turbulent liquid flow during the subsequent sampling process;

[0100] 2) By providing multiple detection areas 5 of different thicknesses, the detection surface formed by the thicker detection areas 5 has a larger sample carrying capacity per unit area, which is suitable for overall and accurate measurement of the number of cells. The detection surface formed by the thinner detection areas 5 has a larger liquid sample spreading area per unit volume, which is suitable for precise differentiation of cell types, thereby achieving accurate measurement of multiple cell parameters in one liquid sample.

[0101] 3) Through the connection and thickness design of the guide groove area 6 and the detection area 5, the liquid sample can flow from the guide groove area 6 to the detection area 5 in a controlled and smooth manner, preventing the liquid front from being disturbed and forming bubbles;

[0102] 4) By adjusting the angle α between the tangent of the downward arc of the sampling port 7 and the horizontal reference plane of the sampling port 7, the liquid sample can be actively sucked into the detection area 5 by capillary force and fill it. By designing the sampling notch 9 of the sampling port 7, the liquid sample can be passively injected into the detection area 5 by capillary force and fill it, thereby achieving compatibility between active suction and passive injection modes.

[0103] 5) The shape, position (in the flow guide area 6 and / or the detection area 5), ​​and number of the exhaust holes 8 can effectively exhaust gas and prevent bubbles in the gas path while preventing the liquid sample from overflowing due to capillary flow inertia. The exhaust holes 8 can be distributed on one side of the sampling and detection cavity 2 or symmetrically arranged on both sides. After sampling, only the side of the sampling and detection cavity 2 needs to be wiped, avoiding loss of liquid sample due to wiping the sampling port.

[0104] 6) By providing a transition fillet 10 on the inner edge of the upper edge of the chip sidewall 4, the liquid at the edge of the sampling and detection cavity 2 can form a stable liquid bridge surface 11, preventing the liquid sample from flowing out due to gravity during the movement of the microfluidic chip.

[0105] Those skilled in the art should understand that these embodiments are only used to illustrate the present invention and do not limit the scope of the present invention. Various equivalent variations and modifications made to the present invention belong to the disclosure of the present invention.

Claims

1. A microfluidic chip for cell counting and typing, characterized in that: The invention comprises a supporting substrate (1) and a sampling detection cavity (2) provided on the supporting substrate (1); the sampling detection cavity (2) is a semi-open cavity formed by two parallel chip side walls (4) with a certain gap, and has a detection area (5), a sampling port (7), and a guide groove area (6) connecting the sampling port (7) and the detection area (5); the thickness H of the detection area (5) is 1 / 4 of the upper and lower portions thereof. 检 Less than the thickness H of the guide groove area (6) 导 The sampling and detection chamber (2) is provided with at least one exhaust hole (8), which is a through hole connecting the interior of the sampling and detection chamber (2) with the outside atmosphere, and which passes through one side wall of the detection area (5) or the guide groove area (6) or symmetrically passes through both side walls of the detection area (5) or the guide groove area (6); The sampling port (7) is provided with a sampling notch (9) for injecting a liquid sample through the sampling notch (9), so that the liquid sample can enter the detection area (5) and fill it by passive capillary injection; the sampling port (7) is in a concave arc shape, and the angle α between the tangent of the downward arc and the horizontal reference plane of the sampling port (7) is in the range of 15° to 45°, so that the liquid sample can enter the detection area (5) and fill it by active absorption through capillary force, thereby realizing the compatibility of active absorption and passive injection modes.

2. The microfluidic chip for cell counting and typing according to claim 1, characterized in that: The exhaust hole (8) is an inverted conical through hole, with the small end of the cone opening facing the inside of the detection area (5) or the guide groove area (6), and the large end of the cone opening facing the outside atmosphere.

3. The microfluidic chip for cell counting and typing according to claim 1, characterized in that: The sampling detection cavity (2) is provided with a detection area (5), and the detection area (5) has a single thickness. ,thickness The range is 60μm-120μm.

4. The microfluidic chip for cell counting and typing according to claim 1, characterized in that: The sampling detection cavity (2) is provided with a plurality of detection areas (5), the plurality of detection areas (5) are independent and connected to each other, and have the same or different thicknesses, and the thickness of any detection area (5) is smaller than the thickness of the guide groove area (6). .

5. The microfluidic chip for cell counting and typing according to claim 4, characterized in that: The sampling detection cavity (2) is provided with two detection areas (5) of different thicknesses, namely a first detection area (51) and a second detection area (52), and the first detection area (51) and the second detection area (52) are connected by the guide groove area (6).

6. The microfluidic chip for cell counting and typing according to any one of claims 1 to 5, characterized in that: The sampling port (7) is located at the upper edge opening of the two chip side walls of the sampling detection cavity (2), wherein the upper edge of one of the two chip side walls (4) located at the sampling port (7) is provided with the sampling notch (9).

7. The microfluidic chip for cell counting and typing according to claim 4 or 5, characterized in that: Each of the detection areas (5) is provided with a corresponding sampling port (7).

8. The microfluidic chip for cell counting and typing according to claim 7, characterized in that: A sampling notch (9) is provided in each of the sampling ports.

9. The microfluidic chip for cell counting and typing according to any one of claims 1 to 5, characterized in that: The inner edge of the chip side wall (4) is provided with a transition fillet (10), and the range of the transition fillet (10) R is 0.2mm-1.5mm.

10. The microfluidic chip for cell counting and typing according to any one of claims 1 to 5, characterized in that: It also includes a sample processing chamber (3) arranged on the supporting substrate (1), which serves as a storage container for the reagent and a mixing operation container for the reagent and the liquid sample to be tested.

11. The microfluidic chip for cell counting and typing according to any one of claims 1 to 5, characterized in that: The support substrate (1) is a handheld portion of the microfluidic chip, and the sampling detection cavity (2) is arranged at the front end of the support substrate (1) and is integrally formed with the support substrate (1) or bonded to the support substrate (1).

12. The microfluidic chip for cell counting and typing according to any one of claims 1 to 5, characterized in that: The detection area (5) is a combination of a rectangle, a square, a trapezoid, a circle or an arc and other shapes, and each shape can be provided with rounded corners, right angles or a combination of rounded corners and right angles.

13. The microfluidic chip for cell counting and typing according to any one of claims 1 to 5, used in conjunction with a detection and analysis instrument for cell counting and typing, characterized in that: The support substrate (1) is further provided with a positioning groove, which is arranged below the support substrate (1) and is used for alignment and positioning with the detection and analysis instrument.

14. The microfluidic chip for cell counting and typing according to any one of claims 1 to 5, characterized in that: Also included are pre-packaged liquid reagents, which are pre-packaged in the following ways: The liquid reagent is added into the sampling detection cavity of the microfluidic chip through the sampling port or sampling notch of the microfluidic chip, and dried or freeze-dried, and the reagent is pre-packaged in the detection cavity; or, The liquid reagent is added into the sample processing chamber of the microfluidic chip and dried or freeze-dried. The reagent is pre-packaged in the sample processing chamber.

15. A sampling method using the microfluidic chip for cell counting and typing according to claim 3, comprising: The sampling port of the microfluidic chip is immersed in a liquid sample for sampling, so that the liquid sample flows from the guide groove area (6) into the detection area (5) under the action of capillary force and fills the detection area (5), wherein the capillary force is proportional to the thickness of the detection area (5). , the thickness of the guide groove area (6) The following relationship is satisfied: 。 16. A sampling method using the microfluidic chip for cell counting and typing according to claim 6, comprising: Immersing the sampling port in a liquid sample for sampling, so that the liquid sample flows from the guide groove area into the detection area under the action of capillary force and fills the detection area; Alternatively, the liquid sample is injected into the sampling detection cavity through the sampling notch for sampling, so that the liquid sample flows from the guide groove area into the detection area and fills the detection area.

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