A microfluidic chip for raw milk cell analysis and a microfluidic sampling device

Abstract

The present invention provides a microfluidic chip for raw milk cell analysis and a microfluidic sampling device for sampling raw milk samples. The microfluidic sampling device includes a bracket assembly and at least one microfluidic chip clamped on the bracket assembly. The microfluidic chip includes a base and a sample injection and detection cavity provided on the base. The sample injection and detection cavity has a sample injection port, an analysis area, and a drainage channel area connecting the sample injection port and the analysis area. The thickness of the analysis area is less than that of the drainage channel area, and a plurality of exhaust holes are provided on the analysis area or the drainage channel area, which can prevent the raw milk sample from blocking the flow path to achieve rapid sample injection of the raw milk sample, avoid the generation of bubbles, realize the precise control of the amount of raw milk cell samples, and simultaneously take into account the precision of cell counting and typing detection, laying a foundation for the on-site precise analysis of raw milk cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical detection, and particularly relates to a microfluidic chip for raw milk cell analysis and a microfluidic sampling device. Background Art

[0002] Somatic cells in raw milk usually include macrophages, lymphocytes, polymorphonuclear neutrophils, and a small amount of mammary tissue epithelial cells, etc. The total number of somatic cells (SSC) contained in each milliliter of raw milk is an index for measuring the quality of raw milk. Taking bovine raw milk as an example, under normal circumstances, there are about 20,000 - 200,000 somatic cells per milliliter of bovine raw milk. Generally speaking, when the lactation system is infected and damaged (such as being invaded by bacteria), the white blood cell count will increase significantly to 500,000 / ml to 1,000,000 / ml, or even higher. The concentration of somatic cells is closely related to livestock management and food safety. For example, in the livestock industry, the concentration of somatic cells can directly reflect the udder health of dairy cows, potential milk production capacity, etc.; in terms of food safety, the concentration of somatic cells is closely related to the composition, quality, and flavor of milk. Therefore, rapid and accurate counting of raw milk somatic cells is helpful for livestock health management and milk quality monitoring.

[0003] Existing total somatic cell count detection technologies mainly target bovine somatic cells and include indirect methods and direct methods. The indirect method refers to detecting the changes in the physicochemical properties of milk caused by the increase in somatic cells, thereby indirectly reflecting the total number of bovine somatic cells; the direct method directly detects and counts the cells. Common indirect methods include the California Mastitis Test (CMT), Wisconsin Mastitis Test (WMT), viscosity method, etc. These methods all use surfactants to release DNA in cells and then measure the DNA aggregates, micelles, viscosity, etc. The main defect of these methods is that the above physicochemical characteristics are not typical enough, and large errors will occur when the milk is not fresh, the fat content is too high, the temperature difference is too large, etc. The most classic direct method is the manual microscopy method, which has gradually been replaced by flow cytometry due to its excessive dependence on manual experience. Flow cytometry is to dilute and drive the cells pre-stained with fluorescence under the wrapping of sheath fluid, and then sequentially pass through an optical detection system to achieve accurate counting. As the most advanced technology at present, it has a fast detection speed and relatively high accuracy, but the operation process is complex, and the unit price and usage cost of the equipment are both high.

[0004] Regarding the advantages and problems of flow cytometry, the prior art US8906697B2 discloses a new direct detection technique. The entire detection device includes three parts, namely, a piston part for facilitating the suction of milk samples, a flow path part for staining the cells in the milk sample, and a detection part for analyzing the stained cells. During use, the front end of the flow path part is placed into the milk sample, the piston part is pushed to suck the milk sample into the flow path part, and it is mixed with a pre-fixed reagent in the flow path. Finally, the milk sample and the reagent mixture flow into the detection part, and the number and morphology of cells are detected based on the fluorescence signal. Compared with flow cytometry, the most significant advantage of this method is that a simple imaging system can be used to quickly analyze the number and morphology of cells on-site. However, this technique has the following defects:

[0005] (1) Lack of design to prevent bubble interference: Raw milk has a high protein content and is extremely prone to generating bubbles during shaking. Due to the lack of a corresponding exhaust structure design, even tiny bubbles in the detection part will cause large errors in cell counting; larger bubbles in the detection part will form large cavities, resulting in greater analysis deviation;

[0006] (2) Lack of design to prevent blockage of milk samples in the flow path: Raw milk has relatively high fat and protein contents, so it is relatively viscous. At the same time, temperature changes are likely to cause it to coagulate and further increase its viscosity. The relatively thin and long flow path part is extremely prone to blockage;

[0007] (3) Lack of design for fine cell analysis: The detection part usually only has a uniform thickness, so it cannot simultaneously meet the requirements of fine analysis of single cells with a small thickness (small thickness, small depth of field, large spreading area per unit volume of raw milk, suitable for fine analysis of single cells) and accurate measurement of the total number of cells with a large thickness (large thickness, large depth of field, large volume carried per unit area of the detection part, suitable for accurate counting of more cells);

[0008] (4) Lack of design for accurate result review: The somatic cell content in raw milk is extremely low (20,000 / ml - 200,000 / ml), and it is extremely prone to large sampling and detection deviations. The lack of result review will affect the accuracy and reliability of the detection results. Summary of the Invention

[0009] To solve the above problems, the present invention provides an improved microfluidic chip for analyzing raw milk cells.

[0010] The technical solution adopted by the present invention is as follows:

[0011] A microfluidic chip for raw milk cell analysis, comprising a base (1) and a sampling and detection chamber (2) provided on the base (1). The sampling and detection chamber (2) is a semi-open cavity formed by two parallel chip side walls (3) with a certain gap, having an analysis area (4), a sampling inlet (5), and a drainage channel area (7) connecting the sampling inlet (5) and the analysis area (4). The thickness H of the analysis area (4) 分 is less than the thickness H of the drainage channel area (7) 引 . The sampling inlet (5) is located at the upper edge opening of the two chip side walls (3) of the sampling and detection chamber (2). One of the two chamber side walls (3) is provided with a sampling notch (8) at the upper edge at the sampling inlet (5) to inject raw milk samples through the sampling notch (8). A notch is opened on the side of the sampling inlet (5) close to the base at the closed end of the sampling and detection chamber (2) as a ventilation groove (9), and the bottom of the ventilation groove (9) is not lower than the bottom of the sampling notch (8).

[0012] In the above microfluidic chip for raw milk cell analysis, at least one exhaust hole (6) is provided on the sampling and detection chamber (2). The exhaust hole (6) is a through hole connecting the inside of the sampling and detection chamber (2) with the outside atmosphere, and it penetrates one side wall of the analysis area (4) or the drainage channel area (7) or symmetrically penetrates both side walls of the analysis area (4) or the drainage channel area (7).

[0013] In the above microfluidic chip for raw milk cell analysis, the exhaust hole (6) is an inverted conical through hole, with the small end of the cone opening towards the inside of the analysis area (4) or the drainage channel area (7), and the large end of the cone opening towards the outside atmosphere.

[0014] In the above microfluidic chip for raw milk cell analysis, there is one analysis area (4) in the sampling and detection chamber (2). The total unilateral area of the analysis area (4) accounts for 50%-90% of the total unilateral area of the sampling and detection chamber (2), and the analysis area (4) has a single thickness H 分 , the thickness H 分 ranges from 50 μm to 400 μm.

[0015] In the above microfluidic chip for raw milk cell analysis, the sampling and detection chamber (2) is provided with multiple analysis areas (4). The total unilateral area of the analysis areas (4) accounts for 50%-90% of the total unilateral area of the sampling and detection chamber (2); the multiple analysis areas (4) are independent of each other and connected, with the same or different thicknesses. The thickness of any analysis area (4) is less than the thickness H of the drainage channel area (7) 引 , the thickness range of any analysis area (4) is 50 μm to 400 μm, and the thickness H of the drainage channel area (7) 引 ranges from 120 μm to 500 μm.

[0016] In the above-mentioned microfluidic chip for raw milk cell analysis, the sample injection and detection chamber (2) is provided with two analysis regions (4) of different thicknesses, namely a first analysis region (41) and a second analysis region (42), and the first analysis region (41) and the second analysis region (42) are connected by a drainage channel region (7).

[0017] In the above-mentioned microfluidic chip for raw milk cell analysis, the sample inlet (5) is in a concave arc shape, and the included angle α between the tangent of its downward-sliding arc and the horizontal reference plane of the sample inlet (5) ranges from 15° to 85°, preferably from 45° to 85°, and more preferably 80°.

[0018] In the above-mentioned microfluidic chip for raw milk cell analysis, each analysis region is provided with a corresponding sample inlet.

[0019] In the above-mentioned microfluidic chip for raw milk cell analysis, each sample inlet is provided with a sample injection notch.

[0020] In the above-mentioned microfluidic chip for raw milk cell analysis, the inner edge of the end of the chip side wall (3) is provided with a transition fillet (10), and the range of the transition fillet R is 0.2 mm - 1.5 mm.

[0021] In the above-mentioned microfluidic chip for raw milk cell analysis, the analysis region (4) is in the shape of a rectangle, square, trapezoid, circle or a combination of an arc and other shapes, and each shape can be provided with fillets, right angles or a combination of fillets and right angles.

[0022] The present invention also provides a microfluidic sampling device for raw milk cell analysis, which is used in cooperation with a detection and analysis instrument for cell counting and typing, and includes a bracket assembly (200) and at least one of the above-mentioned microfluidic chips (100) installed on the bracket assembly (200); preferably, two microfluidic chips (100) are arranged in parallel on the bracket assembly (200).

[0023] In the above-mentioned microfluidic sampling device for raw milk cell analysis, the bracket assembly (200) is provided with a clamping portion (201) for clamping the microfluidic chip (100).

[0024] In the above-mentioned microfluidic sampling device for raw milk cell analysis, the microfluidic chip (100) is provided with an inner positioning groove (13), which is arranged below the base (1) and cooperates with the protrusion on the bracket assembly (200) to achieve accurate assembly and positioning of the microfluidic chip.

[0025] The above-mentioned microfluidic sampling device for raw milk cell analysis is also provided with an outer positioning groove (14), and the outer positioning groove (14) is arranged on the bottom surface of the tail end of the bracket assembly (200) and matches the protrusion on the bearing table in the detection and analysis instrument to achieve precise positioning of the microfluidic sampling device.

[0026] In the above microfluidic sampling device for raw milk cell analysis, a reagent chamber (12) is provided on the bracket assembly (200) for storing reagents and serving as a mixing container for reagents and raw milk samples.

[0027] In the above microfluidic sampling device for raw milk cell analysis, the reagent chamber (12) is cylindrical, frustum-shaped or hemispherical, with a volume of 30 μL - 100 μL and a depth of 5 mm - 14 mm.

[0028] 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 chamber. The sampling and detection chamber has a sampling port, an analysis area, and a drainage groove area connecting the sampling port and the analysis area. The thickness of the analysis area is less than that of the drainage groove area, and a plurality of exhaust holes are provided on the drainage groove area and / or the analysis area. A notch is provided on one side of the sampling port close to the base as a ventilation groove. By setting a sampling notch, a ventilation groove, exhaust holes, and one or a plurality of analysis areas with different thicknesses, the microfluidic chip can prevent the raw milk sample from blocking the flow path to achieve rapid sampling of the raw milk sample, avoid the generation of bubbles, achieve precise control of the amount of raw milk cell samples, and simultaneously take into account the precision of cell counting and typing detection, laying a foundation for the on-site accurate analysis of raw milk cells. Description of the Drawings

[0029] Figure 1A is a schematic plan view of Embodiment 1 of the microfluidic chip of the present invention;

[0030] Figure 1B Figure 1A left view of;

[0031] Figure 1C is a schematic three-dimensional view of Embodiment 1 of the microfluidic chip of the present invention;

[0032] Figure 1D is Figure 1A cross-sectional view taken along line A-A in;

[0033] Figure 2A is a schematic plan view of Embodiment 2 of the microfluidic chip of the present invention;

[0034] Figure 2B is a schematic three-dimensional view of Embodiment 2 of the microfluidic chip of the present invention;

[0035] Figure 2C is Figure 2A cross-sectional view taken along line C1-C1 in;

[0036] Figure 2D is Figure 2A cross-sectional view taken along line C2-C2 in;

[0037] Figure 3AIt is a schematic plan view of the third embodiment of the microfluidic chip of the present invention;

[0038] Figure 3B It is a schematic three-dimensional structure view of the third embodiment of the microfluidic chip of the present invention;

[0039] Figure 3C It is a schematic partial structure view of the sample injection and detection cavity of the third embodiment of the microfluidic chip of the present invention;

[0040] Figure 4A It is a schematic plan view of the first implementation manner of the microfluidic sampling device of the present invention;

[0041] Figure 4B It is a schematic three-dimensional structure view of the first implementation manner of the microfluidic sampling device of the present invention;

[0042] Figure 4C It is Figure 4A the cross-sectional view taken along line B-B in

[0043] Figure 5 It is a schematic plan view of the second implementation manner of the microfluidic sampling device of the present invention;

[0044] Figure 6 It is a schematic three-dimensional structure view of the third implementation manner of the microfluidic sampling device of the present invention;

[0045] Figure 7 It is a schematic three-dimensional structure view of the fourth implementation manner of the microfluidic sampling device of the present invention.

[0046] The reference signs in the figure are shown as:

[0047] 100 - microfluidic chip;

[0048] 200 - bracket assembly, 201 - clamping portion;

[0049] 1 - base; 2 - sample injection and detection cavity; 3 - chip side wall;

[0050] 4 - analysis area, 41 - first analysis area, 42 - second analysis area;

[0051] 5 - sample injection port, 51 - first sample injection port, 52 - second sample injection port;

[0052] 6 - exhaust hole, 61 - first exhaust hole, 62 - second exhaust hole, 63 - third exhaust hole, 64 - fourth exhaust hole;

[0053] 7 - drainage trough area;

[0054] 8 - sample injection notch, 81 - first sample injection notch, 82 - second sample injection notch;

[0055] 9 - ventilation trough;

[0056] 10 - Transition fillet, 11 - Liquid bridge surface, 12 - Reagent chamber;

[0057] 13 - Inner positioning groove; 14 - Outer positioning groove. Specific embodiments

[0058] In order to solve the problems existing in the existing raw milk cell analysis methods, such as the inability to simultaneously meet the requirements of fine single - cell analysis and accurate measurement of the total cell count, the detection part is prone to bubble interference, the milk sample is prone to blockage in the flow path, and the lack of review of detection results, etc., the present invention provides a microfluidic chip and a microfluidic sampling device for raw milk cell analysis. The microfluidic sampling device includes a bracket assembly and at least one microfluidic chip arranged on the bracket assembly. The microfluidic chip includes a base and a sampling and detection cavity arranged on the base. The sampling and detection cavity is a semi - open cavity formed by two parallel chip side walls with a certain gap, having a design of one or multiple analysis areas with different thicknesses, a drainage groove area design for connecting the sampling port and the analysis area, a ventilation groove and an exhaust hole design for removing bubbles. By setting one or multiple analysis areas with different thicknesses, a sampling notch, a ventilation groove and an exhaust hole in the sampling and detection cavity, the microfluidic chip can prevent the viscous milk sample from blocking the flow path, so as to realize the rapid and smooth sampling of the raw milk sample, avoid the generation of bubbles, realize the precise control of the cell sample volume, and simultaneously take into account the precision of cell counting and typing detection, laying a foundation for the on - site precise analysis of raw milk cells.

[0059] The following will describe in detail the microfluidic chip and the microfluidic sampling device for raw milk cell analysis of the present invention with reference to Embodiment 1 to Embodiment 3 and the accompanying drawings.

[0060] Example 1

[0061] Figures 1A - 1D This is a structural example of the microfluidic chip of the present invention. Figures 1A - 1D In the shown Embodiment 1, the microfluidic chip 100 includes a base 1 and a sampling and detection cavity 2 arranged on the base 1. Among them, the tail end of the base 1 is the handheld part of the microfluidic chip 100, and its design is a shape suitable for holding. For example, in this Embodiment 1, the handheld part is rectangular, and the sampling and detection cavity 2 with an arc - shaped edge extends from the front end of the base 1; the sampling and detection cavity 2 can be integrally formed with the base 1, or the sampling and detection cavity 2 is bonded to the front end of the base 1. The sampling and detection cavity 2 is a semi - open cavity formed by two parallel chip side walls 3 with a certain gap, including a sampling port 5, a sampling notch 8, an analysis area 4, an exhaust hole 6, and a drainage groove area 7 connecting the sampling port 5 and the analysis area 4, where:

[0062] In this embodiment, the sample inlet 5 is located at the upper edge openings of the two chip side walls 3 of the sampling and detection cavity 2. The upper edges of the two chip side walls 3 at the sample inlet 5 are provided with sample injection notches 8, which are convenient for positioning the raw milk sample liquid suction pipe, so that the sample can be injected through the sample injection notch 8 by injection. Since the fat and protein contents in raw milk are relatively high, it is relatively viscous, and capillary force alone is not sufficient to suck the raw milk sample into the sampling and detection cavity 2. In the present invention, the raw milk sample is injected into the sampling and detection cavity 2 through the sample injection notch 8 by injection. Under the combined action of the injection power of the liquid suction pipe and the capillary force, the raw milk sample smoothly enters the sampling and detection cavity 2 until it fills the analysis area 4. In order to prevent larger bubbles in the raw milk sample from entering the sampling and detection cavity 2, a notch is opened on the side of the closed end of the sampling and detection cavity 2 near the substrate 1 (the unopened side) of the sample inlet 5 as a ventilation groove 9. The thickness of the ventilation groove 9 is the same as the thickness of the drainage groove area 7, and the bottom of the ventilation groove 9 does not cross the bottom of the sample injection notch 8( Figure 1A is defined as the bottom). When the raw milk sample in the liquid suction pipe is injected through the sample injection notch 8, the larger bubbles in the raw milk sample escape from the ventilation groove 9 on the side of the sample inlet 5, thereby preventing the bubbles from entering the sampling and detection cavity 2.

[0063] The analysis area 4 is located in the sampling and detection cavity 2. The shape of the analysis area 4 can be rectangular, square, trapezoidal, circular or a combination of an arc and other shapes, and 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 analysis area 4; the analysis area 4 has a single thickness H 分 , and the raw milk sample enters the analysis area 4 to form a detection surface. For the analysis area 4 with a large thickness, the sample carrying capacity per unit area of the detection surface is large and the depth of field is large, which is suitable for the overall accurate measurement of the number of raw milk cells; for a small thickness, the spreading area of the same volume of liquid sample on the detection surface is large, which is suitable for the precise differentiation of raw milk cell types. The thickness of the analysis area 4 is 50μm - 400μm.

[0064] The total unilateral area of the analysis area 4 accounts for 50% - 90% of the total unilateral area of the sampling and detection cavity 2. The analysis area 4 has a large area ratio, which can simultaneously increase the raw milk cell carrying capacity and the spreading area, taking into account the overall accurate counting of cells and the precise analysis of single cells; at the same time, the chip side wall 3 at the analysis area 4 is thicker, so that the larger area of the chip side wall 3 is not easily deformed, thereby ensuring that the analysis area 4 has a uniform thickness.

[0065] The drainage groove area 7 is located in the sampling and detection cavity 2 and is connected to the sample inlet 5 and the analysis area 4. The thickness H 引 of the drainage groove area 7 generally ranges from 120μm to 500μm. As Figure 1D shown, the thickness H 分 of the analysis area 4 is less than the thickness H 引, the raw milk sample enters from the sample inlet 5 and is uniformly and rapidly introduced into the analysis area 4 through the flow path formed by the drainage tank area 7 and filled up.

[0066] In this embodiment, the sample inlet 5 is in a concave arc shape, and the tangent of its downward arc ( Figure 1A the left arc in the figure) and the horizontal reference plane of the sample inlet 5 form an angle α (see Figure 1A ), which can determine the flow direction of the raw milk sample to be measured entering the drainage tank area 7, so as to ensure that the raw milk sample to be measured spontaneously flows into the analysis area 4 and is filled in a predetermined manner. The range of the angle α is 15° to 85°. Considering that the raw milk sample has a high fat and protein content and a relatively high viscosity, the angle α is preferably selected from 45° to 85°, and more preferably 80°. Among them, the larger the angle α, the faster the injection rate of the sample, but the greater the probability of the front surface of the sample deforming and generating bubbles. Therefore, 80° is preferably selected.

[0067] The thickness H of the analysis area 4 分 is smaller than the thickness H of the drainage tank area 7 引 , which is beneficial to discharging bubbles. To ensure that the liquid can continuously flow from the drainage tank area 7 into the analysis area 4 and be filled under the action of capillary force, it is required that the capillary pressure is greater than zero. The capillary force has the following relationship with the thickness H of the analysis area 4 分 , the thickness H of the drainage tank area 7 引 , the surface tension of the liquid to be detected, and the contact angle of the liquid to be detected on the surface of the material of the drainage tank area 7:

[0068]

[0069] It can be seen from Equation (1) that by designing the thickness H of the drainage tank area 7 引 and the thickness H of the analysis area 4 分 , the adjustment of different liquid flow rates and liquid laminar flow characteristics can be realized, so as to avoid the formation of bubbles.

[0070] Specifically, in this embodiment, there is one analysis area 4, which is located in the sample injection and detection cavity 2 and has a single thickness H 分 , and its shape is a rounded rectangle. When the thickness H of the analysis area 4 分 is relatively large, the depth of field of the detection surface formed on the chip side wall 3 is large and the sample loading capacity per unit area is large, which is suitable for the overall accurate calculation of the number of raw milk cells. The thickness of the analysis area 4 is preferably 100 μm - 400 μm; when the thickness H of the analysis area 4 分 is relatively small, the depth of field of the detection surface formed on the chip side wall 3 is small and the spreading area of the sample per unit volume is large, which is suitable for the fine analysis of single raw milk cells. At this time, the thickness of the analysis area 4 is preferably 50 μm - 100 μm.

[0071] In order to further avoid the generation of air bubbles, at least one exhaust hole 6 is provided on the sample injection and detection chamber 2. The exhaust hole 6 is a through hole that connects the inside of the sample injection and detection chamber 2 to the outside atmosphere. It can be located on one or both sides of the analysis area 4 or the drainage groove area 7, and can be a symmetric or asymmetric through hole, that is, the exhaust hole 6 penetrates one side wall of the analysis area 4 or the drainage groove area 7 or symmetrically penetrates both side walls of the analysis area 4 or the drainage groove area 7. Preferably, the exhaust hole 6 is a symmetric inverted conical through hole, that is, the small end of the cone opens towards the inside of the drainage groove area 7, and the large end of the cone opens towards the outside atmosphere (see Figure 1D ).

[0072] The advantages of the inverted conical exhaust hole are as follows. First, by using the surface tension between the raw milk sample to be measured and the gas, it is easier to discharge air 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 air bubbles; in the case where reagents need to be pre-encapsulated in the sample injection and detection chamber 2, since the contact surface between the reagent and the outside environment is very small, when the reagent is added to the sample injection and detection chamber 2, its drying process is long. The design of the exhaust hole 6 can increase the contact surface between the reagent and the outside environment, accelerate the drying and uniform distribution of the reagent, thereby avoiding the generation of air bubbles in various situations; it helps to accurately control the sample volume. After the raw milk sample to be measured enters the exhaust hole 6, it is not easy to overflow due to the action of surface tension; the exhaust hole 6 is arranged on one or both sides of the sample injection and detection chamber 2. After the sample injection is completed, only the side of the chip needs to be wiped, avoiding the loss of liquid samples due to wiping the sample injection port; at the same time, the inverted cone hole structure further reduces the possibility of sample wiping loss.

[0073] Specifically, in this embodiment, a first exhaust hole 61 and a second exhaust hole 62 are respectively arranged at the positions of the raw milk sample in the drainage groove area 7 before and after flowing into the analysis area 4. Both exhaust holes are symmetric inverted conical holes that penetrate the side wall 3 of the chip, so as to effectively prevent the residual of air bubbles in the analysis area 4 before and after the raw milk sample flows in.

[0074] Both side walls 3 of the chip can be used for subsequent cell counting and type monitoring analysis. In order to prevent the raw milk sample to be measured from flowing out due to the action of gravity when moving the microfluidic chip, a transition fillet 10 is provided on the inner edge of the end of the side wall 3 of the chip. The range of the transition fillet R is 0.2 mm - 1.5 mm. When the microfluidic chip is injected with samples, a stable liquid bridge surface 11 is formed at the transition fillet 10 at the end of the two side walls 3 of the chip, which can effectively balance the gravity of the liquid to be measured and prevent it from flowing out.

[0075] Specifically, as Figure 1DAs shown, there are two exhaust holes 6, namely a first exhaust hole 61 and a second exhaust hole 62, which are respectively located at the front end (the position before the raw milk sample to be tested enters the analysis area 4) and the rear end (the position after the raw milk sample to be tested flows out of the analysis area 4) of the drainage groove area 7, and are symmetrical inverted conical through holes penetrating the drainage groove area 7, which can further avoid the generation of bubbles.

[0076] In the structural design of the first embodiment, an analysis area 4 with a single thickness can be selected according to the application occasion, and a suitable thickness H can be set. 分 so as to perform high-precision measurement of the total number of cells or fine analysis of single cells.

[0077] Obviously, in this embodiment, an analysis area 4 can also have multiple thicknesses H. 分1 、H 分2 etc., and each thickness value of the analysis area 4 is less than the thickness of the drainage groove area 7. The capillary force driving the liquid sample to be tested into the sampling and detection cavity 2 is related to each thickness of the analysis area 4 and the thickness H of the drainage groove area. 引 still satisfies Equation (1). When the sample enters the analysis area 4, detection surfaces with different depths of field and different spreading states are formed, which can take into account both the overall counting of raw milk cells and the fine analysis of single cells.

[0078] The bracket assembly 200 is provided with a clamping portion 201 for clamping the microfluidic chip 100. The bracket assembly 200 and at least one microfluidic chip 100 clamped in the clamping portion 201 of the bracket assembly 200 form the microfluidic sampling device of the present invention. Figure 5 In the shown embodiment, the microfluidic sampling device includes one microfluidic chip 100, and the microfluidic chip 100 is snapped into any clamping portion 201 of the bracket assembly 200. Figures 4A to 4C In the shown embodiment, the microfluidic sampling device includes two identical microfluidic chips 100, and the microfluidic chips 100 are respectively snapped into the two clamping portions 201 of the bracket assembly 200. The design of multiple microfluidic chips 100 can, on the one hand, accurately review the detection results of each microfluidic chip 100 (that is, detect the two microfluidic chips 100 in sequence, and if the difference between the two detection results does not exceed the preset threshold, it is valid, and the sum or average of the two detection results is taken as the final detection result). On the other hand, since the fluorescence of raw milk cells is weak, the detection results of multiple microfluidic chips 100 can be superimposed to ensure the accuracy of detection.

[0079] Obviously, the maximum number of microfluidic chips 100 in the microfluidic sampling device is determined by the number of clamping portions 201 of the bracket assembly 200. Preferably, the clamping portions 201 on the bracket assembly 200 are arranged in parallel on the same horizontal plane.

[0080] For the convenience of the pretreatment of raw milk samples, a reagent chamber 12 is provided on the bracket assembly 200, which is used for storing solid or liquid reagents and serving as a mixing container for the reagents and raw milk samples. In one embodiment, the reagent chamber 12 is cylindrical, frustum-shaped or hemispherical, with a volume of 30 μL - 100 μL and a depth of 5 mm - 14 mm.

[0081] For the convenience of assembling and clamping with the clamping portion 201 of the bracket assembly 200, an inner positioning groove 13 is further provided on the microfluidic chip 100. The inner positioning groove is arranged below the base 1 of the microfluidic chip, and is matched with the protrusion on the bracket assembly 200 to achieve the accurate assembly and positioning of the microfluidic chip.

[0082] For the convenience of use in subsequent detection and analysis instruments for raw milk cell counting and typing, an outer positioning groove 14 is further provided on the microfluidic sampling device. The outer positioning groove 14 is arranged on the bottom surface of the tail end of the bracket assembly 200, and is used to position the microfluidic sampling device in the detection and analysis instrument. When the microfluidic sampling device is placed in the detection and analysis instrument, the outer positioning groove 14 will match with the protrusion on the carrier table in the detection and analysis instrument, so as to achieve the precise positioning function.

[0083] Example 2

[0084] Figures 2A - 2D The structure of the second embodiment of the microfluidic chip of the present invention is shown. The structure of the second embodiment is a further improvement on the basis of the structure of the first embodiment. The difference between its structure and that of the first embodiment lies in:

[0085] In this embodiment, two analysis areas 4 are provided in the sampling and detection chamber 2, which are respectively the first analysis area 41 and the second analysis area 42. The two analysis areas are independent of each other (set at intervals), and the two analysis areas are connected by a drainage groove area 7. In this embodiment, both analysis areas are rectangular, with thicknesses of H 分1 、H 分2 , where, the thickness H 分1 of the first analysis area 41 is large. The depth of field of the detection surface formed by the side wall 3 of the chip in the first analysis area 41 for the raw milk sample to be measured is large, and the sample carrying capacity per unit area is large, which is suitable for the overall accurate measurement of the raw milk cell number. The thickness H 分1 is preferably 100 μm - 400 μm; the thickness H 分2 of the second analysis area 42 is small. The depth of field of the detection surface formed by the side wall 3 of the chip in the second analysis area 42 for the raw milk sample to be measured is small, and the spreading area of the sample per unit volume is large, which can be used for the fine analysis of raw milk cells. The thickness H 分2 of the second analysis area 42 is preferably 50 μm - 100 μm. This embodiment can take into account both the overall cell counting and the fine analysis of single cells at the same time. The two analysis areas 4 are connected by a drainage groove area 7 with the same thickness.

[0086] Specifically, in this embodiment, there are four exhaust holes 6, namely a first exhaust hole 61, a second exhaust hole 62, a third exhaust hole 63, and a fourth exhaust hole 64. Among them, the first exhaust hole 61 and the third exhaust hole 63 are respectively located at the front end of the drainage groove area 7 (the position before the raw milk sample to be tested enters the first analysis area 41) and the middle part (the position after the raw milk sample to be tested flows out of the first analysis area 41). The second exhaust hole 62 is arranged on the first analysis area 41, and the fourth exhaust hole 64 is arranged on the second analysis area 42. The exhaust holes 6 are all symmetrical inverted conical through holes penetrating the drainage groove area 7 or the analysis area 4, which can further avoid the generation of bubbles.

[0087] Obviously, multiple analysis areas 4 can also have the same thickness, and this thickness value is less than the thickness value of the drainage groove area 7. By setting different analysis areas 4, it is helpful to analyze the accuracy and consistency of the detection results of each analysis area 4. The other structures of the microfluidic chip in Embodiment 2 are the same as those in Embodiment 1 and will not be elaborated.

[0088] The microfluidic chip 100 in this embodiment is assembled with the bracket assembly 200 as a whole to form a microfluidic sampling device, and this microfluidic sampling device includes at least one microfluidic chip 100. As Figure 6 The shown microfluidic sampling device includes two microfluidic chips 100, and the other structures are the same as those in Embodiment 1 and will not be elaborated.

[0089] In the structural design of this Embodiment 2, two analysis areas 4 with different thicknesses respectively correspond to different requirements for total cell counting and single-cell fine analysis.

[0090] Example 3

[0091] Figures 3A - 3C The structure of Embodiment 3 of the microfluidic chip of the present invention is shown. The structure of Embodiment 3 is a further improvement on the basis of the structure of Embodiment 2, and the difference from the structure of Embodiment 2 lies in:

[0092] In this embodiment, there are two sample inlets 5, namely a first sample inlet 51 and a second sample inlet 52, which respectively correspond to the first analysis area 41 and the second analysis area 42. The first sample inlet 51 is provided with a first sample inlet notch 81, and a ventilation groove 9 is opened on one side of the first sample inlet 51 close to the base 1. The second sample inlet 52 is located at the middle part of the opening of the sample injection detection cavity 2; the included angle α between the tangent of the downward arc of the sample inlet 5 and the horizontal reference plane of the sampling port 7 is 15° to 85°, so as to promote the rapid and smooth flow of the viscous raw milk sample into the analysis area 4 and fill it, while avoiding the entry of bubbles. The design of the two sample inlets can prevent the situation that the raw milk sample in the second analysis area 42 at the end of the flow cannot be filled when the liquid is too viscous.

[0093] Specifically, multiple drainage groove areas 7 can be provided (drainage groove areas that are interconnected and have different thicknesses are called different drainage groove areas). Each drainage groove area 7 corresponds to a corresponding analysis area 4, facilitating the controlled and rapid inflow of raw milk samples into the corresponding analysis area 4. By providing drainage groove areas 7 and analysis areas 4 with different thicknesses, the adjustment of different liquid flow rates and liquid laminar flow characteristics can be achieved. The thickness of the analysis area 4 is less than the thickness of all drainage groove areas 7. Similarly, the drainage groove area 7 can be provided as one (a drainage groove area with the same thickness and being interconnected is called the same drainage groove area), and multiple analysis areas 4 can correspond to the same drainage groove area 7.

[0094] Specifically, by adjusting the angle α between the tangent of the downward arc of the sample inlet 5 and the horizontal reference plane of the sample inlet 5, the specific orientation of the raw milk sample to be tested entering the drainage groove area 7 can be specified, so as to ensure that the raw milk sample to be tested flows into the analysis area and fills it in a specific manner. The preferred range of the angle α is 15° to 85°.

[0095] Similarly, the microfluidic chip 100 of Embodiment 3 is snap - connected in the snap - connection part 201 of the bracket assembly 200 to form a microfluidic sampling device, as Figure 7 shown. Two completely identical microfluidic chips 100 are snap - connected in parallel on the bracket assembly 200, which can achieve accurate verification of the detection results to enhance the credibility of the detection results.

[0096] The other structures of Embodiment 3 are the same as those of Embodiment 2. For the technical solutions not mentioned in this Embodiment 3, please refer to Embodiment 2 or Embodiment 1, and will not be elaborated here.

[0097] Based on the above embodiments, further, the material of the microfluidic chip of the present invention can be selected from any one or a combination of optical - grade transparent polymers, glass, and quartz, ensuring high light transmittance and low fluorescence self - emission performance.

[0098] Obviously, the structures of the microfluidic chip and the microfluidic sampling device of the present invention are not limited to the structures described in the above embodiments. Based on the concept of the present invention, by simply increasing or decreasing the number of analysis areas, drainage groove areas, sample inlets (with sample inlet notches), exhaust holes, and snap - connection parts, changing the shape, position, or combination form, they all fall within the scope of the concept of the present invention.

[0099] The method of adding samples to the above - mentioned microfluidic chip: Passive injection based on a pipette, that is, use a pipette to suck the raw milk sample or the raw milk sample to be tested after pretreatment, place the pipette at the sample inlet notch 8 of the sample inlet 5 of the microfluidic chip, and add the raw milk sample to the microfluidic chip until the analysis area 4 is full.

[0100] After adding the samples, the microfluidic chip is snap - connected to the snap - connection part of the microfluidic sampling device, and the microfluidic sampling device is placed at a predetermined position of the detection and analysis instrument to perform detection and analysis.

[0101] The microfluidic chip and microfluidic sampling device for raw milk cell analysis in the above embodiments have the following outstanding features and effects:

[0102] 1) The sample injection and detection chamber 2 of the present invention adopts a semi-open cavity structure. In the case of reagent pre-encapsulation, a smooth gas path accelerates the speed and uniformity of reagent encapsulation, preventing the formation of bubbles due to disordered liquid flow during subsequent sample injection;

[0103] 2) A ventilation groove 9 is provided on the side of the sample injection port 5 close to the base 1. During the sample addition process, large bubbles in the raw milk sample are prevented from entering the sample injection and detection chamber 2, avoiding large detection errors;

[0104] 3) By setting multiple analysis regions 4 with different thicknesses, the detection surface formed by the analysis region 4 with a large thickness has a large sample carrying capacity per unit area, which is suitable for the overall accurate measurement of the number of raw milk cells. The detection surface formed by the analysis region 4 with a small thickness has a large spreading area per unit volume of liquid sample, which is suitable for the precise differentiation of cell types, thus realizing the simultaneous accurate measurement of multiple cell parameters for the same raw milk sample;

[0105] 4) Through the connection and thickness design of the drainage groove region 7 and the analysis region 4 and the design that the total area of the analysis region accounts for 50%-90% of the total area of one side of the sample injection and detection chamber, the controllable and smooth flow of the liquid sample from the drainage groove region 7 to the analysis region 4 is realized, preventing the formation of bubbles due to the disorder of the liquid front, so that the side wall 3 of the chip with a large area is not easily deformed, thus ensuring a uniform thickness in each analysis region;

[0106] 5) In view of the relatively viscous characteristics of raw milk, by setting the angle α between the tangent of the downward arc of the sample injection port 5 and the horizontal reference plane of the sample injection port 5 in a large range of 15° to 85°, the speed and smoothness of the raw milk sample flowing into the sample injection and detection chamber 2 are accelerated;

[0107] 6) Through the design of the shape, position (drainage groove region 7 and / or analysis region 4), and quantity of the exhaust holes 6, while effectively discharging gas through a smooth gas path to prevent bubbles, the liquid sample can be prevented from overflowing due to the capillary flow inertial force; through the design of the distribution of the exhaust holes 6, the exhaust holes 6 are distributed on one side of the sample injection and detection chamber 2 or symmetrically arranged on both sides. After sampling, only the side of the sample injection and detection chamber 2 needs to be wiped, avoiding the loss of the liquid sample due to wiping the sample injection port;

[0108] 7) By providing a transition fillet 10 at the inner edge of the end of the chip side wall 3, a stable liquid bridge surface 11 can be formed at the end of the sample injection and detection chamber 2, avoiding the outflow of the liquid sample due to gravity during the movement of the microfluidic chip;

[0109] 8) Assemble multiple microfluidic chips 100 together through the bracket assembly 200 for simultaneous detection, so as to realize the re-verification analysis of the same raw milk sample in one detection, enhancing the reliability of the detection results.

[0110] 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. All equivalent variations and modifications made to the present invention fall within the scope of the disclosure of the present invention.

Claims

1. A microfluidic chip for raw milk cell analysis, characterized in that, It includes a base (1) and a sample injection and detection cavity (2) provided on the base (1). The sample injection and detection cavity (2) is a semi-open cavity formed by two parallel chip side walls (3) with a certain gap, and has an analysis area (4), a sample injection port (5), and a drainage groove area (7) connecting the sample injection port (5) and the analysis area (4). The thickness H of the analysis area (4) 分 is less than the thickness H of the drainage groove area (7) 引 , the sample injection port (5) is located at the upper edge opening of the two chip side walls (3) of the sample injection and detection cavity (2). One of the two chamber side walls (3) is provided with a sample injection notch (8) at the upper edge at the sample injection port (5) to inject raw milk samples through the sample injection notch (8); a notch is opened on the side of the sample injection port (5) close to the base at the closed end of the sample injection and detection cavity (2) as a ventilation groove (9), and the bottom of the ventilation groove (9) is not lower than the bottom of the sample injection notch (8); The sample injection and detection chamber (2) is provided with a plurality of analysis regions (4), and the thickness of any analysis region (4) is less than the thickness H of the drainage groove region (7). 引 ; The analysis area (4) is in the shape of a rectangle, square, trapezoid, circle, or a combination of an arc and other shapes, and each shape is provided with rounded corners, right angles, or a combination of rounded corners and right angles.

2. The microfluidic chip for raw milk cell analysis according to claim 1, characterized in that At least one exhaust hole (6) is provided on the sample injection and detection chamber (2). The exhaust hole (6) is a through-hole that connects the inside of the sample injection and detection chamber (2) to the outside atmosphere, and it penetrates one side wall of the analysis area (4) or the drainage groove area (7), or symmetrically penetrates both side walls of the analysis area (4) or the drainage groove area (7).

3. The microfluidic chip for raw milk cell analysis according to claim 2, wherein The exhaust hole (6) is an inverted conical through-hole, with the small end of the cone opening towards the inside of the analysis area (4) or the drainage groove area (7), and the large end of the cone opening towards the outside atmosphere.

4. The microfluidic chip for raw milk cell analysis according to any one of claims 1 to 3, characterized in that, An analysis area (4) is provided in the sample injection and detection cavity (2). The total area of one side of the analysis area (4) accounts for 50%-90% of the total area of one side of the sample injection and detection cavity (2), and the analysis area (4) has a single thickness H 分 , and the thickness H 分 ranges from 50 μm to 400 μm.

5. The microfluidic chip for raw milk cell analysis according to any one of claims 1 to 3, characterized in that The sample injection and detection chamber (2) is provided with a plurality of analysis regions (4), and the total area of one side of the analysis regions (4) accounts for 50%-90% of the total area of one side of the sample injection and detection chamber (2); the plurality of analysis regions (4) are independent of each other and connected, and have the same or different thicknesses, and the thickness range of any one analysis region (4) is 50 μm-400 μm, and the thickness H 引 of the drainage groove region (7) ranges from 120 μm to 500 μm.

6. The microfluidic chip for raw milk cell analysis according to claim 5, wherein, The sample injection and detection chamber (2) is provided with two analysis areas (4) of different thicknesses, namely the first analysis area (41) and the second analysis area (42), and the first analysis area (41) and the second analysis area (42) are connected by a drainage groove area (7).

7. The microfluidic chip for raw milk cell analysis according to any one of claims 1 to 3, characterized in that, The sample injection port (5) is in a concave arc shape, and the angle α between the tangent of the downward arc of the sample injection port (5) and the horizontal reference plane of the sample injection port (5) ranges from 15° to 85°.

8. The microfluidic chip for raw milk cell analysis according to claim 5, characterized in that Each analysis area is provided with a corresponding sample injection port.

9. The microfluidic chip for raw milk cell analysis according to claim 8, wherein Each sample injection port is provided with a sample injection notch.

10. The microfluidic chip for raw milk cell analysis according to any one of claims 1 to 3, characterized in that, The inner edge of the end of the chip side wall (3) is provided with a transition rounded corner (10), and the range of the transition rounded corner R is 0.2 mm - 1.5 mm.

11. A microfluidic sampling device for raw milk cell analysis, which is used in conjunction with a detection and analysis instrument for cell counting and typing, is characterized in that, It includes a bracket assembly (200) and at least one microfluidic chip (100) according to any one of claims 1 to 10 mounted on the bracket assembly (200); two microfluidic chips (100) are arranged in parallel on the bracket assembly (200).

12. The microfluidic sampling device for raw milk cell analysis according to claim 11, wherein, The bracket assembly (200) is provided with a clamping portion (201) for clamping the microfluidic chip (100).

13. The microfluidic sampling device for raw milk cell analysis according to claim 12, wherein An inner positioning groove (13) is provided on the microfluidic chip (100). The inner positioning groove is arranged below the base (1) and cooperates with the protrusion on the bracket assembly (200) to achieve accurate assembly and positioning of the microfluidic chip.

14. The microfluidic sampling device for raw milk cell analysis according to claim 12 or 13, characterized in that, An outer positioning groove (14) is also provided. The outer positioning groove (14) is arranged on the bottom surface at the end of the bracket assembly (200) and matches the protrusion on the bearing platform in the detection and analysis instrument to achieve precise positioning of the microfluidic sampling device.

15. The microfluidic sampling device for raw milk cell analysis according to claim 12 or 13, characterized in that, A reagent chamber (12) is provided on the bracket assembly (200) for storing reagents and serving as a mixing container for reagents and raw milk samples.

16. The microfluidic sampling device for raw milk cell analysis according to claim 15, characterized in that, The reagent chamber (12) is in the shape of a cylinder, an inverted frustum of a cone, or a hemisphere, with a volume of 30 μL - 100 μL and a depth of 5 mm - 14 mm.

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