Integrated cell counting chip and cell staining and counting method

Abstract

The invention provides an integrated cell counting chip and a cell staining and counting method. The integrated cell counting chip comprises a body, and the cell counting chip further comprises a clamping interface, a cell counting cavity, a sample inlet and a cell staining reagent which are located in the body; a first flow channel is arranged between the sample inlet and the cell counting cavity, and a second flow channel is connected between the clamping interface and the cell counting cavity; according to the cell counting chip, the clamping interface, the cell counting cavity and the sample inlet are integrated in the chip, and the cell staining reagent is arranged in the chip, so that when an external negative pressure source device sucks a cell suspension sample through the cell counting chip, the cell suspension flows into the cell counting chip to dissolve the cell staining reagent, and staining is completed; and then the cell counting cavity is matched with the external imaging device to complete counting, so that the cell counting operation can be completed only by sucking the sample once, the cell counting steps are simplified, and the detection flux and efficiency of cell counting are improved.

Description

Technical Field

[0001] The invention relates to a counting chip, in particular to an integrated cell counting chip, and belongs to the technical field of biomedical detection. Background Art

[0002] Cell counting is a very important detection method and tool, which is mainly used to perform statistical analysis on the number of human cells, various bacteria, fungi and even some specific particles, to obtain parameters such as the concentration and viability of various cell suspensions, and to provide researchers or clinicians with status information of the human body and biological reaction systems.

[0003] Currently, there are two methods for cell staining and counting: manual counting and instrument-automated counting. The counting process and steps are roughly the same, and the difference is the final counting method. Manual counting uses the naked eye, while instrument-automated counting uses a computer to collect images and obtain results after processing. Taking trypan blue as an example, the detection steps of the two are:

[0004] Step 1: Prepare the staining reagent, usually dilute the trypan blue into a solution of about 0.4%;

[0005] Step 2: staining, adding 0.4% trypan blue solution to the cell suspension, and making the final concentration of trypan blue in the cell suspension about 0.04%;

[0006] Step 3: Counting: Add the stained cell suspension to the cell counting chip, and use a microscopic imaging device such as a microscope to obtain the cell concentration and viability by naked eye or computer processing.

[0007] Among them, step 1 and step 2 require manual pipetting several times, and use corresponding consumables such as centrifuge tubes and pipette tips; and in step 3, not only manual pipetting is required, but also a dedicated cell counting chip is required. From step 1 to step 3, it is not difficult to see that the existing process of manual counting or automatic cell counting is quite cumbersome and complicated, and the consumables involved include cell counting chips, multiple pipette tips and centrifuge tubes, etc., which are costly and time-consuming, and are contrary to the high-throughput automation of current medical testing, which hinders the development of high-throughput testing in the field of cell counting. For this reason, an integrated cell counting chip and cell staining and counting method are proposed. Summary of the invention

[0008] In view of this, and thanks to the development of microfluidic chip technology, it is possible to achieve quantification, transportation, and mixing of fluids inside a microfluidic chip. The present invention provides an integrated cell counting chip and a cell staining and counting method, which abandons the cumbersome process of steps 1 to 3 in the prior art to solve or alleviate the technical problems existing in the prior art, and at least provide a beneficial choice.

[0009] The technical solution of the embodiment of the present invention is implemented as follows: an integrated cell counting chip includes a body, the cell counting chip also includes a clamping interface, a cell counting cavity, an injection port and a cell staining reagent located in the body;

[0010] Wherein, a first flow channel is provided between the sample inlet and the cell counting chamber, a second flow channel is connected between the clamping interface and the cell counting chamber, and the cell staining reagent is encapsulated inside the body;

[0011] Wherein, the sample inlet is used to absorb the cell sample deeply into the cell suspension;

[0012] Wherein, the first flow channel is used to transport the cell sample sucked from the injection port into the cell counting chamber;

[0013] Wherein, the cell counting chamber is used to contain a cell suspension and is connected to an external imaging device for cell imaging;

[0014] Wherein, the second flow channel is used to transmit air;

[0015] Wherein, the clamping interface is used for connecting an external negative pressure device with a cell counting chip.

[0016] Further preferably, the outer diameter of the body at the clamping interface is less than 9.5 mm, and the maximum width in the radial direction is less than 9.5 mm.

[0017] Further preferably, the cell counting chamber is a flat chamber with a depth range of 10 μm-300 μm and an area range of 1 mm 2 -300mm 2 ;

[0018] Wherein, the shape of the cell counting chamber includes but is not limited to circular and rectangular;

[0019] Both sides of the cell counting chamber are optically transparent surfaces.

[0020] Further preferably, the cell staining reagent includes but is not limited to trypan blue and AO / PI staining reagent; the packaging method of the cell staining reagent includes but is not limited to air drying and freeze drying.

[0021] Further preferably, the cell counting chip further comprises a reagent chamber;

[0022] Wherein, the reagent cavity is located between the first flow channel and the cell counting cavity, and the cell staining reagent is located inside the reagent cavity;

[0023] The depth of the reagent cavity is 10 μm-500 μm and the area is 1 mm 2 -200mm 2;

[0024] The cell staining reagent is enclosed in a reagent chamber within the body.

[0025] Further preferably, the cell counting chip further comprises one or more mixing channels, wherein the mixing channels are connected between the cell counting chamber and the sample inlet, and are used to mix the inhaled cell suspension or the cell suspension and the cell staining reagent;

[0026] Wherein, the depth of the mixing channel is 100 μm-1000 μm;

[0027] The depths of the cell counting cavity and the reagent cavity are both smaller than the depths of the first flow channel, the second flow channel and the mixing flow channel.

[0028] Further preferably, the mixing channel is a planar or three-dimensional structure, and its shape includes but is not limited to a serpentine channel, a channel with local contraction and expansion, and a channel with internal obstacles, and any combination of the three, and the height direction of the combined mixing channel is the same.

[0029] Further preferably, the cell counting chip further comprises a throttle port, a fixing structure and a focusing structure for microscopic imaging, wherein the throttle port is a structure in which the size of the local flow channel increases or decreases sharply, and the throttle port is located between the second flow channel and the clamping interface, and is used to throttle the liquid flowing out of the cell counting cavity to prevent it from entering the clamping interface;

[0030] The fixing structure is arranged outside the body;

[0031] The position of the focusing structure includes but is not limited to the surface of the body and the inner wall of the cell counting chamber, and the shape of the focusing structure includes but is not limited to lines, patterns or a combination of the two.

[0032] Further preferably, the cell counting chip further comprises an air hole, which is opened on the outer side of the body and communicated with the second flow channel, and is used for exhausting air.

[0033] An integrated cell staining and counting method comprises the following steps:

[0034] S1, connecting and fixing the cell counting chip with an external pressure device;

[0035] S2. Driven by an external pressure device, the cell suspension is sucked into the sample inlet with a volume of 10 μL-30 μL;

[0036] S3, the cell suspension enters the reagent chamber through the first flow channel to dissolve the cell staining reagent;

[0037] S4, fully mixing the cell suspension and cell staining reagent flowing through the mixing channel to complete cell staining;

[0038] S5, the stained cell suspension enters the cell counting chamber;

[0039] S6. The cell image information in the cell counting chamber is directly obtained through a microscopic imaging device, and the cell viability is calculated using image recognition technology.

[0040] The embodiment of the present invention adopts the above technical solution, and has the following advantages: the present invention integrates a clamping interface, a cell counting chamber, and a sample injection port inside the chip, and arranges a cell staining reagent inside the chip, so that when an external negative pressure source device absorbs a cell suspension sample through the cell counting chip, the cell suspension flows into the cell counting chip to dissolve the cell staining reagent and complete the staining, and then the cell counting chamber is used in conjunction with an external imaging device to complete the counting, so that the cell counting operation can be completed by only sucking the sample once, thereby simplifying the cell counting steps and improving the cell counting detection throughput and efficiency.

[0041] The above summary is for illustrative purposes only and is not limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0043] Figure 1 It is a structural diagram of the cell counting chip of the present invention;

[0044] Figure 2 It is a schematic diagram of the connection between the cell counting chip of the present invention and a pipette, a syringe pump and a pipette gun;

[0045] Figure 3 It is a detection schematic diagram of the microscopic imaging device of the present invention;

[0046] Figure 4 This is a schematic diagram of the structure in which the cell staining reagent of the present invention is encapsulated in a cell counting chamber;

[0047] Figure 5 It is a schematic diagram of the 2D plane structure of the mixing channel of the present invention;

[0048] Figure 6 This is a schematic diagram of the structure in which the cell staining reagent of the present invention is encapsulated in the reagent cavity;

[0049] Figure 7 It is a schematic diagram of the structure of the sudden flow channel, small cavity and multi-cavity of the present invention;

[0050] Figure 8 It is a schematic diagram of the structure of the pore of the present invention;

[0051] Fig. 9 This is a schematic diagram of the cell counting chip structure according to the first embodiment of the present invention;

[0052] Fig.10 This is a schematic diagram of the cell counting chip structure according to the second embodiment of the present invention;

[0053] Fig.11 FIG. 4 is a schematic diagram of the structure of a cell counting chip according to the third embodiment of the present invention.

[0054] Figure numerals: 1, main body; 101, clamping interface; 102, first flow channel; 103, cell counting chamber; 104, mixing flow channel; 105, injection port; 106, second flow channel; 107, reagent chamber; 108, fixing structure; 109, cell staining reagent; 110, throttle port; 111, focusing structure; 112, pore. DETAILED DESCRIPTION

[0055] In the following, only some exemplary embodiments are briefly described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the present invention. Therefore, the drawings and descriptions are considered to be exemplary and non-restrictive in nature.

[0056] It should be noted that the terms "first", "second", "symmetrical", "array", etc. are only used to distinguish between description and position description purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the definition of "first", "symmetrical", etc. can explicitly or implicitly include one or more of these features; similarly, when the number of certain features is not limited in the form of words such as "two" or "three", it should be noted that this feature also explicitly or implicitly includes one or more feature quantities.

[0057] The embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0058] like Figure 1 As shown, an embodiment of the present invention provides an integrated cell counting chip, including a body 1, the cell counting chip also includes a clamping interface 101, a cell counting chamber 103, an injection port 105 and a cell staining reagent 109 located in the body 1;

[0059] Among them, a first flow channel 102 is provided between the injection port 105 and the cell counting chamber 103, a second flow channel 106 is connected between the clamping interface 101 and the cell counting chamber 103, and a cell staining reagent 109 is encapsulated inside the body 1;

[0060] The sample inlet 105 is used to absorb the cell sample deeply into the cell suspension;

[0061] The first flow channel 102 is used to transport the cell sample sucked by the injection port 105 into the cell counting chamber 103;

[0062] The cell counting chamber 103 is used to contain a cell suspension and is connected to an external imaging device for cell imaging;

[0063] Wherein, the second flow channel 106 is used to transmit air;

[0064] Wherein, the clamping interface 101 is used for connecting the external negative pressure device with the cell counting chip;

[0065] The clamping interface 101 is used to connect an external pressure source device, such as a pipette, a syringe pump, a peristaltic pump, etc., which can drive the counting chip to absorb the cell suspension through negative pressure. The negative pressure source is the most commonly used pressure source. Driven by the external negative pressure source device, the sample enters from the injection port 105, passes through the first flow channel 102, and enters the cell counting chamber 103. Correspondingly, the gas in the first flow channel 102 and the cell counting chamber 103 passes through the second flow channel 106 and the clamping interface 101 and enters the negative pressure device. Figure 2 As shown, different connection modes between the clamping interface 101 and the external pressure device are shown. In this connection mode, the clamping interface 101 is a circular hole-shaped interface, which can cooperate with the cylindrical external surfaces 201, 301 and 401 of the corresponding interface parts of the external pressure device such as the pipette 2, the injection pump 3, the pipette gun 4, etc. to form a seal, so that the flow channel and the cavity inside the cell counting chip are connected with the pressure device.

[0066] It should be noted that the circular clamping interface 101 is only an example.

[0067] In one embodiment, the outer diameter of the body 1 at the clamping interface 101 is less than 9.5 mm, and the maximum width in the radial direction is less than 9.5 mm;

[0068] The outer diameter of the body 1 at the clamping interface 101 is as follows Figure 1 As shown in the outer diameter D, the outer diameter D is less than 9.5 mm, and the maximum width in the radial direction is less than 9.5 mm, so that the cell counting chip can be compatible with existing pipette guns, automated liquid workstations and other instruments for counting various well plates.

[0069] In one embodiment, the cell counting chamber 103 is a flat chamber with a depth range of 10 μm-300 μm and an area range of 1 mm 2 -300mm 2 ;

[0070] The shape of the cell counting chamber 103 includes but is not limited to a circle or a rectangle;

[0071] Both sides of the cell counting chamber 103 are optically transparent surfaces;

[0072] The cell counting chamber 103 uses the optically transparent surfaces on both sides so that the microscopic imaging device 5 can image the cells in the cell counting chamber 103. Figure 3 As shown, the microscopic imaging device 5 generally has functions such as optical magnification, image acquisition, cell image recognition and statistics, and can effectively count the cells in the cell counting chamber 103 to obtain the cell concentration and viability. The microscopic imaging device 5 images the cells in the cell counting chamber 103 from the optically transparent surface.

[0073] In one embodiment, the cell staining reagent 109 includes but is not limited to trypan blue and AO / PI staining reagent; the packaging method of the cell staining reagent 109 includes but is not limited to air drying and freeze drying;

[0074] The function of the cell staining reagent 109 is to stain the cells and distinguish dead cells from live cells according to the different cell staining results, and then count the proportion of live cells; the conventional cell staining step usually requires a pipette to separately aspirate the staining reagent and the cell suspension, and to mix them by suction and spitting in a vessel such as a centrifuge tube. The whole process requires multiple pipetting and replacement of the gun tip, and the steps are cumbersome and time-consuming. The cell staining reagent 109 is placed inside the body 1. After the cell suspension enters the chip, it will automatically mix with the cell staining reagent 109 and stain the cells, which simplifies the cell counting detection steps and improves efficiency and throughput. The packaging position of the cell staining reagent 109 can be inside the cell counting chamber 103, as shown in FIG. Figure 4 shown.

[0075] In one embodiment, the cell counting chip further includes one or more mixing channels 104, which are connected between the cell counting chamber 103 and the sample inlet 105, and are used to mix the inhaled cell suspension or the cell suspension and the cell staining reagent 109 to ensure random distribution of the cell or particle sample in the suspension;

[0076] Wherein, the depth of the mixing channel 104 is 100 μm-1000 μm;

[0077] The depths of the cell counting chamber 103 and the reagent chamber 107 are both smaller than the depths of the first flow channel 102 , the second flow channel 106 , and the mixing flow channel 104 ;

[0078] When the microscopic imaging device 5 images the cell counting chamber 103, the imaging area is generally 0.2 mm 2 -4 mm 2 , is to image a very small part of the original sample. Therefore, when the sample is unevenly distributed, the accuracy of cell counting will be greatly reduced. Using the mixing channel 104 can improve the statistical accuracy of cell counting.

[0079] The mixing channel 104 is a channel with a 2D planar structure, and its shape includes but is not limited to a serpentine channel 1041, a serpentine channel with local expansion 1042, a straight channel with contraction and expansion 1043, a channel with contraction and expansion and an obstruction inside 1044, and a more complex asymmetric mixing channel 1045, as shown in FIG. Figure 5 As shown, the combined mixing channels 104 are the same in height direction. The processing method of this channel is relatively simple and compatible with the processing technology of the cell counting chip, thereby reducing the production cost.

[0080] The mixing channel 104 may also have a 3D structure. The mixing channel 104 with a 3D structure has a more sufficient mixing efficiency, and a more evenly distributed cell suspension can be obtained in the cell counting chamber 103, thereby improving the detection accuracy.

[0081] In one embodiment, the cell counting chip further includes a reagent chamber 107;

[0082] The reagent chamber 107 is located between the first flow channel 102 and the cell counting chamber 103 , and the cell staining reagent 109 is located inside the reagent chamber 107 ;

[0083] The depth of the reagent cavity 107 is 10 μm-500 μm and the area is 1 mm 2 -200mm 2 ;

[0084] The shape of the reagent chamber 107 includes, but is not limited to, circular, oval, and square;

[0085] At this time, the cell staining reagent 109 is packaged into the reagent cavity 107, such as Figure 6As shown, after the cell suspension passes through the injection port 105 and the first flow channel 102, it reaches the reagent chamber 107, and is mixed with the cell staining reagent 109 in the reagent chamber 107, and is further mixed through the mixing flow channel 104 to ensure that the cells and the cell staining reagent 109 are fully mixed and stained. The stained cell suspension enters the cell counting chamber 103, is collected by the microscopic imaging device 5, and the viability is calculated. The efficiency and staining effect of cell staining can be effectively improved, which is beneficial to improving the statistical accuracy of cell counting and viability.

[0086] In one embodiment, the cell counting chip further includes a throttle port 110, a fixing structure 108 and a focusing structure 111 for microscopic imaging. The throttle port 110 is a structure in which the size of a local flow channel increases or decreases sharply. The throttle port 110 is located between the second flow channel 106 and the clamping interface 101, and is used to throttle the liquid flowing out of the cell counting chamber 103 to prevent it from entering the clamping interface 101.

[0087] The fixing structure 108 is disposed outside the body 1;

[0088] The position of the focusing structure 111 includes but is not limited to the surface of the body 1 and the inner wall of the cell counting chamber 103 , and the shape of the focusing structure 111 includes but is not limited to lines, patterns, or a combination of the two;

[0089] Most cells are transparent, so it is difficult to focus on them. When the focusing structure 111 is used, the speed and accuracy of cell focusing can be increased.

[0090] By setting a fixing structure 108, the cell counting chip can be placed in the well plate rack, which is convenient for loading the pipette and the liquid workstation;

[0091] By setting the throttle port 110, in this rapidly changing structure, the contact angle, surface tension and capillary force will play a leading role, thereby increasing the driving pressure required for the fluid to flow through the throttle port 110. When the throttle port 110 is provided, when the sample sucked from the injection port 105 reaches the throttle port 110, the sample will stay at this position and will not enter the external negative pressure source device;

[0092] The cell counting chip is a microfluidic chip, and the flow channel and cavity volume thereof are small, so the fluid it controls is small, but the flow channel resistance is large, that is, the required driving pressure is large, which poses a challenge to the control performance of the external negative pressure device. By adding the throttle port 110, the control accuracy requirement of the external negative pressure device can be effectively reduced;

[0093] The throttle port 110 may be a sudden change flow channel 1101, or may be a small cavity 1102 or a multi-cavity 1103 composed of a plurality of micro-cavities connected together. Figure 7shown.

[0094] In one embodiment, the cell counting chip further includes an air hole 112, which is opened on the outer side of the body 1 and communicates with the second flow channel 106, and the air hole 112 is used for exhausting air;

[0095] The cell suspension is sucked by the capillary force rather than an external pressure source through the pore 112. In this case, the pore 112 is used to discharge the excess gas in the cell counting chip. Figure 8 shown.

[0096] Embodiment 1

[0097] like Fig. 9 As shown, an embodiment of the present invention provides an integrated cell counting chip, including an injection port 105, a first flow channel 102, a reagent chamber 107, a mixing flow channel 104, a cell counting chamber 103, a second flow channel 106, a fixing structure 108, a cell staining reagent 109, a focusing structure 111 and a clamping interface 101;

[0098] Among them, the cell staining reagent 109 is an AO / PI staining reagent, which is packaged in the reagent chamber 107 by air drying. The cell suspension passes through the injection port 105, passes through the first flow channel 102, and enters the reagent chamber 107; after being mixed with the AO / PI staining reagent in the reagent chamber 107, it enters the mixing flow channel 104. After passing through the mixing flow channel 104, the cells are fully mixed with the AO / PI staining reagent and then enter the cell counting chamber 103. The microscopic imaging device images the cells in the cell counting chamber 103 and obtains the concentration and viability parameters.

[0099] Embodiment 2

[0100] like Fig.10 As shown, the embodiment of the present invention also provides an integrated cell counting chip, including an injection port 105, a first flow channel 102, a cell counting chamber 103, a second flow channel 106, a fixing structure 108, a cell staining reagent 109, a focusing structure 111 and a clamping interface 101. The cell staining reagent 109 is a trypan blue staining reagent, which is packaged in the cell counting chamber 103 by air drying.

[0101] The cell suspension passes through the injection port 105, passes through the first flow channel 102, and enters the cell counting chamber 103, where it is mixed with the trypan blue staining reagent and the cells are stained.

[0102] The chip also has a focusing structure 111, which is in the shape of a cross and is located on the surface of the cell counting chip. It is prepared by silk-screen printing and has a depth of 10 μm. The microscopic imaging device first focuses on the focusing structure 111, obtains the chip position parameters, and then moves to the cell counting cavity 103 for focusing. The chip surface and the location of the cell counting cavity 103 have a fixed distance of 0.5 mm. Therefore, after the focusing structure 111 is focused, it needs to move 0.5 mm to achieve focusing on the cells in the cell counting cavity 103.

[0103] The cell counting chip has a length L=80 mm, an outer diameter 8 of the clamping interface 101 is mm, a cell counting cavity 103 has a depth of 200 μm, a width of 5 mm, and a length of 7 mm. The cross-sectional dimensions of the first flow channel 102 and the second flow channel 106 are 0.8 mm×0.6 mm.

[0104] Embodiment 3

[0105] like Fig.11 As shown, an embodiment of the present invention further provides an integrated cell counting chip, including an injection port 105, a first flow channel 102, a reagent chamber 107, a mixing flow channel 104, a cell counting chamber 103, a second flow channel 106, a fixing structure 108, a cell staining reagent 109, a focusing structure 111 and a clamping interface 101;

[0106] The cell staining reagent 109 is an AO / PI staining reagent, which is packaged in the reagent cavity 107 by air drying.

[0107] The cell suspension passes through the injection port 105, passes through the first flow channel 102, and enters the reagent chamber 107. After being mixed with the AO / PI staining reagent in the reagent chamber 107, it enters the mixing flow channel 104. After passing through the mixing flow channel 104, the cells and the staining reagent are fully mixed, and then enter the cell counting chamber 103. The microscopic imaging device images the cells in the cell counting chamber 103 and obtains the concentration and viability parameters.

[0108] The reagent cavity 107 is rectangular in shape, with a length of 5 mm, a width of 2.5 mm, and a depth of 0.2 mm.

[0109] The mixing channel 104 is a commonly used serpentine channel, which forms a Dean secondary flow inside the channel through multiple turns, thereby improving the mixing efficiency of cells and staining reagents. The cross-sectional size of the serpentine channel is 0.8 mm×0.4 mm.

[0110] The chip also has a focusing structure 111, which is in the shape of a cross and is located on the surface of the cell counting chip. It is prepared by silk-screen printing and has a depth of 10 μm. The microscopic imaging device first focuses on the focusing structure 111, obtains the chip position parameters, and then moves to the cell counting cavity 103 for focusing. The chip surface and the location of the cell counting cavity 103 have a fixed distance of 0.5 mm, so after the focusing structure 111 is focused, it needs to move 0.5 mm to achieve focusing on the cells in the cell counting cavity 103.

[0111] The cell counting chip has a length L=80 mm, an outer diameter of the clamping interface 101 is 8 mm, a depth of the cell counting cavity 103 is 200 μm, a width of 5 mm, and a length of 7 mm. The cross-sectional dimensions of the first flow channel 102 and the second flow channel 106 are 0.8 mm×0.6 mm.

[0112] Embodiment 4

[0113] Based on Example 1, Example 2 or Example 3, the embodiment of the present invention further provides an integrated cell staining and counting method, comprising the following steps:

[0114] S1, connecting and fixing the cell counting chip with an external pressure device using 11;

[0115] S2. Under the drive of the external pressure device, the cell suspension is sucked into the sample inlet 105, with a volume of 10 μL-30 μL;

[0116] S3, the cell suspension enters the reagent chamber 107 through the first flow channel 102 to dissolve the cell staining reagent 109;

[0117] S4, fully mixing the cell suspension and the cell staining reagent 109 flowing through the mixing channel 104 to complete cell staining;

[0118] S5, the stained cell suspension enters the cell counting chamber 103;

[0119] S6. Directly obtain cell image information in the cell counting chamber 103 through a microscopic imaging device, and calculate cell viability using image recognition technology.

[0120] The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of various changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. An integrated cell counting chip, comprising a body (1), characterized in that: The cell counting chip further comprises a clamping interface (101), a cell counting chamber (103), an injection port (105) and a cell staining reagent (109) located in the body (1); Wherein, a first flow channel (102) is provided between the sample inlet (105) and the cell counting chamber (103), a second flow channel (106) is connected between the clamping interface (101) and the cell counting chamber (103), and the cell staining reagent (109) is encapsulated inside the body (1); Wherein, the sample inlet (105) is used to absorb the cell sample deeply into the cell suspension; Wherein, the first flow channel (102) is used to transport the cell sample sucked by the injection port (105) into the cell counting chamber (103); Wherein, the cell counting chamber (103) is used to contain a cell suspension and is connected to an external imaging device for cell imaging; Wherein, the second flow channel (106) is used to transmit air; Wherein, the clamping interface (101) is used to connect an external negative pressure device to the cell counting chip.

2. The integrated cell counting chip according to claim 1, characterized in that: The outer diameter of the body (1) at the clamping interface (101) is less than 9.5 mm, and the maximum width in the radial direction is less than 9.5 mm.

3. The integrated cell counting chip according to claim 1, characterized in that: The cell counting chamber (103) is a flat chamber with a depth range of 10 μm-300 μm and an area range of 1 mm 2 -300mm 2 ; Wherein, the shape of the cell counting chamber (103) includes but is not limited to a circle or a rectangle; Both sides of the cell counting chamber (103) are optically transparent surfaces.

4. The integrated cell counting chip according to claim 1, characterized in that: The cell staining reagent (109) includes but is not limited to trypan blue and AO / PI staining reagent; the packaging method of the cell staining reagent (109) includes but is not limited to air drying and freeze drying.

5. The integrated cell counting chip according to claim 4, characterized in that: The cell counting chip also includes a reagent chamber (107); Wherein, the reagent chamber (107) is located between the first flow channel (102) and the cell counting chamber (103), and the cell staining reagent (109) is located inside the reagent chamber (107); The reagent cavity (107) has a depth of 10 μm-500 μm and an area of ​​1 mm 2 -200mm 2 .

6. The integrated cell counting chip according to claim 4, characterized in that: The cell counting chip further comprises one or more mixing channels (104), wherein the mixing channels (104) are connected between the cell counting chamber (103) and the sample inlet (105) and are used to mix the inhaled cell suspension or the cell suspension and the cell staining reagent (109); Wherein, the depth of the mixing channel (104) is 100 μm-1000 μm; The depths of the cell counting chamber (103) and the reagent chamber (107) are both smaller than the depths of the first flow channel (102), the second flow channel (106) and the mixing flow channel (104).

7. The integrated cell counting chip according to claim 6, characterized in that: The mixing channel (104) is a planar or three-dimensional structure, and its shape includes but is not limited to a serpentine channel, a channel with local contraction and expansion, and a channel with internal obstacles. Any combination of the three, the combined mixing channel (104) is the same in height direction.

8. The integrated cell counting chip according to claim 1, characterized in that: The cell counting chip further comprises a throttle port (110), a fixing structure (108) and a focusing structure (111) for microscopic imaging. The throttle port (110) is a structure in which the size of a local flow channel increases or decreases sharply. The throttle port (110) is located between the second flow channel (106) and the clamping interface (101) and is used to throttle the liquid flowing out of the cell counting chamber (103) to prevent the liquid from entering the clamping interface (101). The fixing structure (108) is arranged on the outside of the body (1); The position of the focusing structure (111) includes but is not limited to the surface of the body (1) and the inner wall of the cell counting chamber (103), and the shape of the focusing structure (111) includes but is not limited to a line, a pattern or a combination of the two.

9. The integrated cell counting chip according to claim 1, characterized in that: The cell counting chip further comprises an air hole (112), wherein the air hole (112) is arranged on the outside of the body (1) and is connected to the second flow channel (106), and the air hole (112) is used for exhausting air.

10. An integrated cell staining and counting method, characterized in that: The following steps are involved: S1, connecting and fixing the cell counting chip with an external pressure device using (11); S2. Under the drive of the external pressure device, the cell suspension is sucked into the sample inlet (105), with a volume of 10 μL-30 μL; S3, the cell suspension enters the reagent chamber (107) through the first flow channel (102) to dissolve the cell staining reagent (109); S4, fully mixing the cell suspension and the cell staining reagent (109) flowing through the mixing channel (104) to complete cell staining; S5, the stained cell suspension enters the cell counting chamber (103); S6. Directly obtain cell image information in the cell counting chamber (103) through a microscopic imaging device, and calculate cell viability using image recognition technology.

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