A microfluidic detection chip

Abstract

The application discloses a microfluidic detection chip, which has a rotation center and comprises one or more detection units; the detection unit comprises a quantitative distribution microchannel arranged around the rotation center, a first liquid storage pool arranged in the quantitative distribution microchannel, a first waste liquid pool communicated with the end of the quantitative distribution microchannel, and a plurality of reaction units arranged along the quantitative distribution microchannel; each first liquid storage pool is connected with the quantitative distribution microchannel through a first siphon valve and is provided with a sealable sample adding hole; the reaction unit comprises a second liquid storage pool arranged in the inside of the quantitative distribution microchannel, a metering pool, a reaction pool and a second waste liquid pool arranged outside the quantitative distribution microchannel; the metering pool is connected with the reaction pool through a second siphon valve, and the second waste liquid pool is connected with the second siphon valve through an air path balance microchannel; the reaction pool is connected with the outside through an air hole. The application can automatically complete the steps of sequential release, quantitative distribution, metering collection, mixing reaction and waste liquid removal of reagents on the chip, and repeatedly execute these steps.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microfluidic technology, and more particularly relates to a microfluidic detection chip. BACKGROUND

[0002] As a new scientific technology, microfluidic technology has been applied in many fields such as chemistry, biology, engineering and physics, and has strong interdisciplinary characteristics. It has made breakthroughs in the precise control of time, space and analysis objects, and can solve many key problems of life analysis. Microfluidic technology can integrate detection experiments that can only be completed in a laboratory onto a small chip, not only saving material and time costs, but more importantly, it can integrate multiple detection technologies into one, improving detection efficiency. However, a complete biochemical reaction generally requires various samples and reagents to be added to the reaction tube in sequence by a pipette for mixing reaction. More complex reaction processes require intermediate removal of liquid and addition of new reagents and samples, and finally signal detection. Therefore, the sequence release of reagents, metering of liquid, mixing reaction, removal of waste liquid, cycle repetition and finally signal acquisition are a complete biochemical analysis detection process. At present, most chip systems based on microfluidic technology can only complete one to three functions of automation, and need to be combined with expensive external intelligent equipment.

[0003] Microfluidic technology applied to in vitro sample analysis and detection can be applied to highly integrated analysis and detection of nucleic acids, proteins, enzymes, sugars and other analytes in samples, and has a wide range of application scenarios in various fields of analysis and detection. For example, in the patent document with application number 202110825892, a chip for microchannel-assisted high-throughput reagent quantitative distribution and analysis is provided, which optimizes the processing technology of the chip through the design of the microchannel, and combines a portable centrifugal device to complete the quantitative distribution and final mixing collection of three liquids, or even multiple liquids, but to some extent, it does not solve the problem of sequential release of liquid, quantitative collection of waste liquid, and re-collection of liquid for reaction. Therefore, a chip system that can independently complete the sequential release, quantitative distribution, metering collection, mixing reaction, and waste liquid removal of reagents on the chip and can cycle these steps is the key to promoting the application of microfluidic technology in the analysis field. SUMMARY

[0004] In view of the defects of the prior art and the need for improvement, the present application provides a microfluidic detection chip, which aims to improve the structure of the microfluidic chip, so that the sequential release, quantitative distribution, metering collection, mixing reaction, and waste liquid removal of reagents can be automatically completed on the chip, and these steps can be repeated.

[0005] To achieve the above object, the present application provides a microfluidic detection chip with a rotation center, which comprises one or more detection units, wherein each detection unit comprises:

[0006] a quantitative dispensing microchannel arranged in a spiral shape around the rotation center, one or more first liquid storage pools arranged inside the quantitative dispensing microchannel, a plurality of reaction units arranged along the quantitative dispensing microchannel, and a first waste liquid pool connected to the end of the quantitative dispensing microchannel;

[0007] Each first liquid storage pool is connected to the first end of the quantitative dispensing microchannel through a first siphon valve, and a sample addition hole is arranged on the first siphon valve; the bending part at the beginning of the first siphon valve has hydrophilicity;

[0008] The reaction unit comprises a second liquid storage pool arranged inside the quantitative dispensing microchannel, and a metering pool, a reaction pool and a second waste liquid pool arranged in sequence from inside to outside outside the quantitative dispensing microchannel; the second liquid storage pool is connected to the metering pool through a first microchannel, the metering pool is connected to the reaction pool through a second siphon valve, the reaction pool is connected to the second waste liquid pool through a second microchannel, and the second waste liquid pool is connected to the second siphon valve through a gas path balance microchannel; the reaction pool is connected to the outside through a gas hole, the volume of the reaction pool is greater than that of the metering pool, and an independent sample addition hole is arranged on the second liquid storage pool; the bending part at the beginning of the second siphon valve has hydrophilicity.

[0009] Further, the connection point of one of the first liquid storage pools and the first siphon valve is higher than the bottom end thereof.

[0010] Further, the reaction pool is an oval-shaped chamber with two micro-protrusions; the two micro-protrusions are respectively the connection points of the second siphon valve and the gas hole and the reaction pool.

[0011] Further, the metering pool is a square combined with a bottom triangular chamber.

[0012] Further, the second microchannel is a meandering microchannel.

[0013] Further, the first liquid storage pool is a fan ring-shaped chamber.

[0014] Further, the connection point of the gas path balance microchannel and the second siphon valve is the end point of the part of the second siphon valve subjected to hydrophilic treatment.

[0015] Further, the microfluidic detection chip provided by the present application is a multilayer structure, and comprises, from bottom to top, a bottom cover, a siphon valve layer, a microchannel layer, a chamber layer and an upper cover.

[0016] The first siphon valve and the second siphon valve are both located in the siphon valve layer.

[0017] The quantitative distribution microchannel, the first microchannel, the second microchannel and the gas path balance microchannel are located in the microchannel layer;

[0018] The first liquid storage pool, the first waste liquid pool, the second liquid storage pool, the metering pool, the reaction pool and the second waste liquid pool are located in the chamber layer.

[0019] The air hole, the sealable sample adding hole and the independent sample adding hole are located in the upper cover.

[0020] Overall, the above technical solutions conceived by the present application can achieve the following beneficial effects:

[0021] The microfluidic detection chip provided by the present application is provided with a plurality of first liquid storage pools inside the quantitative distribution microchannel, and each first liquid storage pool is provided with a sealable sample adding hole and is connected with the quantitative distribution microchannel through a first siphon valve. When the sealable sample adding hole is in a sealed state, the first liquid storage pool is in a relatively sealed state, and no matter how centrifugation is performed, the liquid in the first liquid storage pool cannot break through the resistance of the siphon valve. When the sealable sample adding hole is connected with the atmosphere, the pressure in the first liquid storage pool is released, and the corresponding siphon valve is automatically triggered, so that the liquid is introduced into the quantitative distribution microchannel through the siphon valve. Based on this design, the microfluidic detection chip provided by the present application can control the release sequence of the liquid in each first liquid storage pool under the cooperation of the sealable sample adding hole and the first siphon valve.

[0022] The microfluidic detection chip provided by the present application is provided with an independent second waste liquid pool in each reaction unit arranged along the quantitative distribution microchannel, and the second waste liquid pool is connected with a second siphon valve connecting the metering pool and the reaction pool through a gas path balance microchannel. During the process of introducing the liquid into the reaction pool through the second siphon valve, the second waste liquid pool is in a relatively sealed state and has a large resistance. Therefore, the reaction pool can collect the liquid at a low speed without entering the second waste liquid pool, and the stable liquid introduction of the liquid in the metering pool can be completed at a low speed by relying on the action of the second siphon valve. After the liquid introduction in the metering pool is completed, the second waste liquid pool is connected with the outside through the gas path balance microchannel, the second siphon valve, the reaction pool and the air hole. In a high-speed centrifugation state, the reacted liquid in the reaction pool will break through the second microchannel connecting the reaction pool and the second waste liquid pool and enter the second waste liquid pool. After the liquid in the reaction pool is completely removed, subsequent reactions can be continuously performed, and each step can be cyclically executed.

[0023] Overall, the microfluidic detection chip provided by the present application can automatically complete the steps of sequential release, quantitative distribution, metering collection, mixing reaction and waste liquid removal of reagents on the chip, and repeatedly execute these steps. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1A microfluidic detection chip schematic diagram provided for the embodiment 1 of the present application;

[0025] Figure 2 A first reservoir distribution schematic diagram provided for the embodiment 1 of the present application;

[0026] Figure 3 Another reaction unit schematic diagram provided for the present application;

[0027] Figure 4 A multi-layer structure schematic diagram of the microfluidic detection chip provided for the embodiment 1 of the present application;

[0028] Figure 5 A simulation process schematic diagram of the microfluidic detection chip provided for the embodiment 1 of the present application for enzyme-linked immunoassay of serum procalcitonin (PCT);

[0029] Figure 6 A result analysis diagram of low concentration sample test provided for the embodiment 1 of the present application;

[0030] Figure 7 A microfluidic detection chip schematic diagram provided for the embodiment 2 of the present application;

[0031] Figure 8 A multi-layer structure schematic diagram of the microfluidic detection chip provided for the embodiment 2 of the present application;

[0032] Figure 9 A pigment verification process schematic diagram of the microfluidic detection chip provided for the embodiment 2 of the present application for single sample multi-target detection of enzyme-linked immunoassay;

[0033] Figure 10 A real reagent verification schematic diagram of the microfluidic detection chip provided for the embodiment 2 of the present application for single sample multi-target detection of enzyme-linked immunoassay;

[0034] In all the drawings, the same reference signs are used to represent the same elements or structures, wherein:

[0035] 1-rotation center;

[0036] 2-quantitative dispensing microchannel;

[0037] 3-first reservoir; 31-sealable sample hole, 32-first siphon valve;

[0038] 4-reaction unit; 41-second reservoir, 42-first microchannel, 43-measuring pool, 44-second siphon valve, 45-reaction pool, 46-second microchannel, 47-second waste pool, 48-gas path balance microchannel, 49-gas hole;

[0039] 5-first waste pool. DETAILED DESCRIPTION

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0041] In this invention, the terms "first," "second," etc. (if present) in the invention and the accompanying drawings are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence.

[0042] To automate the sequential release, quantitative distribution, metered collection, mixing reaction, and waste liquid removal of reagents on a chip and to repeat these steps, this invention provides a microfluidic detection chip. The overall concept is that, through structural improvements, some chambers can be in a relatively sealed state or in a state connected to the atmosphere. The corresponding chambers can be switched between these two states by control, thereby controlling the liquid in the chambers to be guided to the designated chambers in the desired order. The chambers for liquid mixing reaction can be reused.

[0043] It should be noted that the microfluidic detection chip provided by this invention is a centrifugal microfluidic chip with a center of rotation. It is easy to understand that in this type of chip, "inner side" and "outer side" refer to the side closer to the center of rotation and the side farther from the center of rotation, respectively; "top" and "bottom" refer to the end closest to the center of rotation and the end farthest from the center of rotation, respectively; descriptions such as "start," "first end," "last end," "upstream," and "downstream" are determined by referring to the flow direction of the liquid during centrifugation. For example, in a channel, the end through which the liquid first flows is the first end, and the end through which it last flows is the last end; or, for another example, the position through which the liquid first flows is the upstream, and the position through which it flows later is the downstream. In the following embodiments, unless otherwise specified, the meanings of the terms are the same as described herein.

[0044] The following is an example.

[0045] Example 1:

[0046] A microfluidic detection chip having a rotation center 1, such as Figure 1 and Figure 2 As shown, the microfluidic detection chip specifically includes: a detection unit; the detection unit includes:

[0047] A quantitative distribution microchannel 2 is arranged in a spiral shape around the rotation center 1, six first liquid storage tanks 3 are arranged inside the quantitative distribution microchannel 2, multiple reaction units 4 are arranged along the quantitative distribution microchannel 2, and a first waste liquid tank 5 is connected to the end of the quantitative distribution microchannel 2.

[0048] Each first liquid storage tank 3 is connected to the beginning of the quantitative distribution microchannel 2 through a first siphon valve 32 and is provided with a sealable sample addition hole 31; the bend at the beginning of the first siphon valve 32 is hydrophilic.

[0049] The reaction unit 4 includes: a second storage tank 41 disposed inside the quantitative distribution microchannel 2, and a metering tank 43, a reaction tank 45, and a second waste tank 47 disposed outside the quantitative distribution microchannel 2 and arranged sequentially from the inside to the outside; the second storage tank 41 is connected to the metering tank 43 through a first microchannel 42, the metering tank 43 is connected to the reaction tank 45 through a second siphon valve 44, the reaction tank 45 is connected to the second waste tank 47 through a second microchannel 46, and the second waste tank 47 is connected to the second siphon valve 44 through a gas balance microchannel 48; the reaction tank 45 is connected to the outside through a vent 49, and the volume of the reaction tank 45 is larger than the volume of the metering tank 43; the second storage tank 41 is provided with an independent sample addition port; the initial bend of the second siphon valve 44 is hydrophilic.

[0050] The microfluidic detection chip provided in this embodiment has multiple first liquid reservoirs arranged inside the quantitative distribution microchannel. Each first liquid reservoir has a sealable sample dispensing orifice, and each first liquid reservoir is connected to the quantitative distribution microchannel via a first siphon valve. When the sealable sample dispensing orifice is sealed, the first liquid reservoir is in a relatively sealed state, and the liquid cannot break through the siphon valve regardless of centrifugation. When the sealable sample dispensing orifice is open to the atmosphere, the pressure in the first liquid reservoir is released, automatically triggering the corresponding siphon valve, and the liquid is guided into the quantitative distribution microchannel through the siphon valve. Based on this design, the microfluidic detection chip provided in this embodiment can control the release sequence of liquid from each first liquid reservoir through the cooperation of the sealable sample dispensing orifice and the first siphon valve.

[0051] The microfluidic detection chip provided in this embodiment has an independent second waste liquid pool in each reaction unit set along the quantitative distribution microchannel. This waste liquid pool is connected to a second siphon valve connecting the metering pool and the reaction pool via a gas path balancing microchannel. During the process of liquid flowing into the reaction pool through the second siphon valve, the second waste liquid pool is in a relatively sealed state with high resistance. Therefore, the reaction pool can collect liquid at a low speed without it entering the second waste liquid pool. Simultaneously, the liquid in the metering pool can be stably drawn at a low speed thanks to the action of the second siphon valve. After the liquid in the metering pool is drawn out, the second waste liquid pool is connected to the outside environment via the gas path balancing microchannel, the second siphon valve, the reaction pool, and the vent. Under high-speed centrifugation, the reacted liquid in the reaction pool will break through the second microchannel connecting the reaction pool and the second waste liquid pool and enter the second waste liquid pool. After all the liquid in the reaction pool has been removed, subsequent reactions can continue. Based on this, each step can be executed cyclically.

[0052] Overall, the microfluidic detection chip provided in this embodiment can automatically complete steps such as sequential release of reagents, quantitative distribution, metered collection, mixing reaction, and waste liquid removal on the chip, and repeat these steps.

[0053] In practical applications, reagents or samples can be pre-packaged into the first reservoir and sealed after being placed through the sealable sample dispensing port on the first reservoir, or they can be added on-site as needed based on the type of reagent required. The liquid in each first reservoir is distributed to the metering cell of each reaction unit via a quantitative dispensing microchannel. The second reservoir is separate for each reaction unit, and samples or reagents can be added as needed through its independent sample dispensing port. Because each reaction cell has an independent space, different targets or different samples can be detected simultaneously.

[0054] During operation, when the liquid in the first storage tank is released, the siphon valve will be quickly triggered, allowing the sealed liquid to enter the quantitative distribution microchannel and gradually enter each metering tank. Excess liquid will enter the first waste liquid tank under high-speed centrifugation. Subsequently, after control, the liquid in the metering tank will enter the reaction tank. Reagents or samples entering from the sample dispensing port will enter the metering tank under centrifugation and further enter the reaction tank. The liquid after reaction in the reaction tank can be drained to the second waste liquid tank.

[0055] In this embodiment, within each reaction unit, the volume of the reaction tank is larger than that of the metering tank. This causes bubbles to form in the reaction tank after the liquid from the metering tank enters it. During liquid mixing, these bubbles can accelerate the reaction rate. As a preferred embodiment, such as... Figure 1As shown, in this embodiment, the reaction tank 45 is an elliptical chamber with two micro-protrusions; the two micro-protrusions are the connection points of the second siphon valve 44 and the air hole 49 with the reaction tank 45, respectively. This structural design is beneficial for forming bubbles and accelerating the reaction.

[0056] like Figure 1 As shown, in this embodiment, the metering pool 43 is a square combined with a triangular chamber at the bottom. This structural design facilitates the flow of liquid in the metering pool 43 into the second siphon valve 44.

[0057] like Figure 1 As shown, in this embodiment, the second microchannel 46 connecting the reaction tank 45 and the second waste liquid tank 47 is a meandering microchannel. This structural design can increase the resistance of the second microchannel 46, ensuring that the liquid in the reaction tank 45 will not enter the second waste liquid tank 47 before the reaction is completed.

[0058] like Figure 1 and Figure 2 As shown, in this embodiment, the first liquid storage tank 3 is designed as a fan-shaped annular chamber. Based on this structural design, more first liquid storage chambers can be arranged in a limited space, and mutual interference between the first siphon valves can be avoided.

[0059] like Figure 1 As shown, in this embodiment, the connection point between the gas path balancing microchannel 48 and the second siphon valve 44 is the same as the connection point between the second siphon valve 44 and the reaction tank 45. It should be noted that this is only one optional connection method; in practical applications, other methods can be used. Figure 3 Another connection method shown is that the connection point between the gas path balance microchannel 48 and the second siphon valve 44 is the end point of the part of the second siphon valve 44 after hydrophilic treatment. In some other embodiments, the specific connection point can also be between these two points, as long as it allows the second waste liquid pool to switch between two states of relative sealing and communication with the atmosphere.

[0060] Optionally, the microfluidic detection chip provided in this embodiment has a 5-layer structure, such as... Figure 4 As shown, these 5 layers, from bottom to top, include a bottom cover, a siphon valve layer, a microchannel layer, a chamber layer, and a top cover.

[0061] Both the first siphon valve and the second siphon valve are located in the siphon valve layer;

[0062] The quantitative distribution microchannel, the first microchannel, the second microchannel, and the gas path balancing microchannel are all located in the microchannel layer;

[0063] The first liquid storage tank, the first waste liquid tank, the second liquid storage tank, the metering tank, the reaction tank, and the second waste liquid tank are all located in the chamber layer;

[0064] The vent, the sealable sample dispensing port, and the independent sample dispensing port are all located on the top cover.

[0065] like Figure 1 and Figure 3 As shown, to facilitate the flow of liquid from the first reservoir into the first siphon valve, in this embodiment, the connection point between each first reservoir and the first siphon valve is located at the bottom of the first reservoir. The same reagent can be added to each reaction tank through the first reservoir, and different samples can be added through the second reservoir of each reaction unit in this embodiment. Therefore, the microfluidic detection chip provided in this embodiment can be used for the detection of multiple samples against a single target. The following explanation uses the enzyme-linked immunosorbent assay (ELISA) of serum procalcitonin (PCT) as an example.

[0066] For ease of description, the six first reservoirs in the detection unit are named Reservoir A, Reservoir B, Reservoir C, Reservoir D, Reservoir E, and Reservoir F, respectively. Reservoir A is pre-encapsulated with blocking solution, Reservoir B with first washing solution, Reservoir C with second washing solution, Reservoir D with signal-labeled antibody, Reservoir E with third washing solution, and Reservoir F with chromogenic substrate. PCT antibody is pre-embedded in the reaction cell to capture PCT antigen in the sample.

[0067] The corresponding single-target multi-sample detection process includes the following steps:

[0068] 1. High-speed centrifugation prepares all reagents, meaning that when the pressure in the first storage tank is released, the corresponding first siphon valve can be automatically triggered;

[0069] 2. The liquid storage tank A is released by puncturing the membrane of the sealable sample addition hole, which automatically and quickly triggers the first siphon valve. High-speed centrifugation allows the sealed liquid to enter the quantitative distribution microchannel and then the metering cell. Excess liquid will enter the first waste liquid tank under high-speed centrifugation.

[0070] 3. When centrifugation stops, all metering cells automatically trigger the corresponding second siphon valve, and then start low-speed centrifugation to guide the liquid into the reaction tank;

[0071] 4. The reaction tank is connected to the outside world through a vent, so the resistance is very small. The second waste liquid tank, which is connected to the reaction tank, is in a relatively sealed state with greater resistance. Therefore, the reaction tank can collect liquid at a low speed. At the same time, the liquid in the metering tank can be stably drained at a low speed by relying on the characteristics of the siphon valve.

[0072] 5. After the closed liquid is collected in the reaction tank, due to the special design of the chamber, the reaction tank is slightly larger than the metering tank. When the liquids are mixed, microbubbles can be generated to accelerate the reaction rate.

[0073] 6. After the sealing liquid is collected in the reaction tank, a mixing reaction is carried out. It is preferable to choose a reaction time of 1 hour, during which the mixture is shaken back and forth clockwise and counterclockwise using a centrifuge.

[0074] 7. After the reaction is complete, start high-speed centrifugation. At this time, the centrifugal force is greater than the resistance of the second microchannel. The second waste liquid pool is in a state of communication with the outside world under high-speed centrifugation, which further reduces the resistance of the second waste liquid pool and increases the stability of the chip. Therefore, high-speed centrifugation can remove the liquid after the reaction in the reaction pool and guide it into the second waste liquid pool.

[0075] 8. After centrifugation stops, the pressure in storage tank B can be selectively released, automatically triggering the corresponding first siphon valve;

[0076] 9. High-speed centrifugation allows the first cleaning solution to enter the quantitative distribution microchannel and then the metering tank. Excess liquid will enter the first waste liquid tank under high-speed centrifugation.

[0077] 10. Then repeat steps 3-7, optionally prioritizing a reaction time of 1 minute in the reaction tank to clean the reaction tank;

[0078] 11. After centrifugation stops, the sample to be tested can be selectively added to the second storage tank of each reaction unit;

[0079] 12. Start the high-speed centrifugation to allow the sample in the second storage tank to flow into the metering tank through the first microchannel;

[0080] 13. Then repeat steps 3-7, optionally prioritizing a reaction time of 20 minutes in the reaction tank;

[0081] 14. After centrifugation stops, the pressure in the storage tank C can be selectively released, automatically triggering the corresponding first siphon valve;

[0082] 15. High-speed centrifugation allows the second cleaning solution to enter the quantitative distribution microchannel and then the metering cell. Excess liquid will enter the first waste liquid tank under high-speed centrifugation.

[0083] 16. Then repeat steps 3-7, optionally prioritizing a reaction time of 1 minute in the reaction tank to clean the reaction tank;

[0084] 17. After centrifugation stops, the pressure in the storage tank D can be selectively released, automatically triggering the corresponding first siphon valve;

[0085] 18. High-speed centrifugation allows the horseradish peroxidase-labeled anti-PCT antibody to enter the quantitative dispensing microchannel and then into the metering cell. Excess liquid will enter the first waste liquid tank under high-speed centrifugation.

[0086] 19. Then repeat steps 3-7, optionally prioritizing a reaction time of 20 minutes in the reaction tank;

[0087] 20. After centrifugation stops, the pressure in the storage tank E can be selectively released, automatically triggering the corresponding first siphon valve;

[0088] 21. High-speed centrifugation allows the third cleaning solution to enter the quantitative distribution microchannel and then the metering tank. Excess liquid will enter the first waste liquid tank under high-speed centrifugation.

[0089] 22. Then repeat steps 3-7, optionally prioritizing a reaction time of 2 minutes in the reaction tank;

[0090] 23. After centrifugation stops, the pressure in the storage tank F can be selectively released, automatically triggering the corresponding first siphon valve;

[0091] 24. High-speed centrifugation allows the chromogenic substrate of horseradish peroxidase to enter the quantitative distribution microchannel and then into the metering cell. Excess liquid will enter the first waste liquid tank under high-speed centrifugation.

[0092] 25. Then repeat steps 3-7, optionally with a reaction time of 20 minutes in the reaction tank;

[0093] 26. Perform signal acquisition, optionally including image acquisition and grayscale analysis.

[0094] To demonstrate the reliability of the detection chip, this invention simulates the entire process using a pigment solution, such as... Figure 5 As shown in the figure, the results illustrate that the microfluidic detection chip provided by the present invention can effectively realize the detection of multiple samples on a single target.

[0095] Further analysis of the results from the low-concentration sample tests yielded the following results: Figure 6 As shown in the figure, the results indicate that the minimum detection concentration of the microfluidic detection chip provided in this embodiment can reach 0.1 ng / mL.

[0096] It should be noted that in practical applications, the number of detection units contained in the chip, the number of first liquid reservoirs in the units, the number of reaction units, and the number of chip layers can be set according to specific detection requirements. The relevant settings in this embodiment are only an optional setting method and should not be construed as the only limitation of the present invention.

[0097] Example 2:

[0098] A microfluidic detection chip, such as Figure 7 and Figure 8As shown, this embodiment includes two detection units. The structure of each detection unit is similar to that of Embodiment 1. The difference is that in each detection unit, the connection point between the first storage tank and the first siphon valve is higher than its bottom. Based on this structural design, by centrifugation, solid impurities will be deposited at the bottom of the storage tank. After triggering the first siphon valve, the supernatant will be guided to the quantitative distribution microchannel through the first siphon valve, while the solid impurities remain in the storage tank. Therefore, the first storage tank has the function of separating solid impurities, and can complete the separation of impurities and supernatant in the sample. For example, it can complete the separation of blood cells and serum in whole blood.

[0099] In this embodiment, the specific implementation of the remaining structures in the detection unit is the same as in Embodiment 1 above, and can be referred to the description in Embodiment 1 above.

[0100] Optionally, in this embodiment, the chip also has a 5-layer structure, and the structural design of each layer is the same as that in Embodiment 1 above, as described in Embodiment 1 above.

[0101] In this embodiment, the same sample can be added to each reaction unit through the first reservoir with solid-phase impurity separation function, and different reagents can be added through the second reservoir of each reaction unit. Therefore, the microfluidic detection chip provided in this embodiment can be used for single-sample multi-target detection. The following description uses the enzyme-linked immunosorbent assay (ELISA) of serum procalcitonin (PCT), interleukin-6 (IL-6), acute phase reactive protein (CRP), and COVID-19 antibody (IgG) as an example, specifically using one detection unit as an example.

[0102] Similarly, for ease of description, the first reservoir in the detection unit with solid-phase impurity separation function is referred to as the sample reservoir, and the other five first reservoirs are named reservoir A, reservoir B, reservoir C, reservoir D, reservoir E, and reservoir F, respectively. Reservoir A is pre-encapsulated with blocking solution, reservoir B is pre-encapsulated with first cleaning solution, reservoir C is pre-encapsulated with second cleaning solution, reservoir D is pre-encapsulated with third cleaning solution, and reservoir E is pre-encapsulated with horseradish peroxidase chromogenic substrate. The four second reservoirs in the detection unit are pre-encapsulated with horseradish peroxidase-labeled anti-PCT antibody, anti-IL-6 antibody, anti-CRP antibody, and anti-IgG antibody, respectively. In the four reaction cells, one pre-encapsulated PCT antibody captures PCT antigen in the sample, one pre-encapsulated IL-6 antibody captures IL-6 antigen in the sample, one pre-encapsulated CRP antibody captures CRP antigen in the sample, and one pre-encapsulated IgG antibody captures IgG antibody in the sample.

[0103] The corresponding single-sample multi-target detection process includes the following steps:

[0104] 1. High-speed centrifugation prepares all reagents, meaning that when the pressure in the first storage tank is released, the corresponding first siphon valve can be automatically triggered;

[0105] 2. The liquid storage tank A is released by puncturing the membrane of the sealable sample addition hole, which automatically and quickly triggers the first siphon valve and centrifuges at high speed, allowing the sealed liquid to enter the quantitative distribution microchannel and then into the metering cell. Excess liquid will enter the first waste liquid tank under high-speed centrifugation.

[0106] 3. When centrifugation stops, all metering cells automatically trigger the corresponding second siphon valve, and then start low-speed centrifugation to guide the liquid into the reaction tank;

[0107] 4. The reaction tank is connected to the outside world through a vent, so the resistance is very small. The second waste liquid tank, which is connected to the reaction tank, is in a relatively sealed state with greater resistance. Therefore, the reaction tank can collect liquid at a low speed. At the same time, the liquid in the metering tank can be stably diverted at a low speed by relying on the characteristics of the siphon valve.

[0108] 5. After the closed liquid is collected in the reaction tank, due to the special design of the chamber, the reaction tank is slightly larger than the metering tank. When the liquid is mixed, microbubbles can be generated to accelerate the reaction rate.

[0109] 6. After the sealing liquid is collected in the reaction tank, a mixing reaction is carried out. Optionally, the reaction time is set to 1 hour, during which the mixture is shaken back and forth clockwise and counterclockwise by a centrifuge.

[0110] 7. After the reaction is complete, start high-speed centrifugation. At this time, the centrifugal force is greater than the resistance of the second microchannel. The second waste liquid pool is in a state of communication with the outside world under high-speed centrifugation, which further reduces the resistance of the second waste liquid pool and increases the stability of the chip. Therefore, high-speed centrifugation can remove the liquid after the reaction in the reaction pool and guide it into the second waste liquid pool.

[0111] 8. After centrifugation stops, the pressure in storage tank B can be selectively released, automatically triggering the corresponding first siphon valve;

[0112] 9. High-speed centrifugation allows the first cleaning solution to enter the quantitative distribution microchannel and then the metering tank. Excess liquid will enter the first waste liquid tank under high-speed centrifugation.

[0113] 10. Then repeat steps 3-7. Optionally, the reaction time in the reaction tank is set to 1 minute, and the reaction tank is cleaned.

[0114] 11. After centrifugation stops, the sample to be tested can be selectively added to the sample pool;

[0115] 12. If the addition of a sample does not trigger the first siphon valve, start high-speed centrifugation to separate the solid and liquid components in the sample solution, thereby separating impurities from the sample. The first siphon valve will be automatically triggered after centrifugation stops.

[0116] 13. Start the high-speed centrifugation to allow the sample to flow into the metering cell through the microchannel;

[0117] 14. Then repeat steps 3-7, optionally setting the reaction time in the reaction tank to 20 minutes;

[0118] 14. After centrifugation stops, the pressure in the storage tank C can be selectively released, automatically triggering the corresponding first siphon valve;

[0119] 15. High-speed centrifugation allows the second cleaning solution to enter the quantitative distribution microchannel and then the metering cell. Excess liquid will enter the first waste liquid tank under high-speed centrifugation.

[0120] 16. Then repeat steps 3-7. Optionally, set the reaction time in the reaction tank to 1 minute and clean the reaction tank.

[0121] 17. After centrifugation stops, the corresponding signal-labeled antibody can be selectively added to each of the second sample loading chambers;

[0122] 18. High-speed centrifugation allows the horseradish peroxidase-labeled anti-PCT antibody, anti-IL-6 antibody, anti-CRP antibody, and anti-IgG antibody solutions to enter the metering cell;

[0123] 19. Then repeat steps 3-7, optionally setting the reaction time in the reaction tank to 20 minutes;

[0124] 20. After centrifugation stops, the pressure in the storage tank D can be selectively released, automatically triggering the corresponding first siphon valve;

[0125] 21. High-speed centrifugation allows the third cleaning solution to enter the quantitative distribution microchannel and then the metering tank. Excess liquid will enter the first waste liquid tank under high-speed centrifugation.

[0126] 22. Then repeat steps 3-7. Optionally, set the reaction time in the reaction tank to 2 minutes and clean the reaction tank.

[0127] 23. After centrifugation stops, the pressure in the storage tank E can be selectively released, automatically triggering the corresponding first siphon valve;

[0128] 24. High-speed centrifugation allows the chromogenic substrate of horseradish peroxidase to enter the quantitative distribution microchannel and then into the metering cell. Excess liquid will enter the first waste liquid tank under high-speed centrifugation.

[0129] 25. Then repeat steps 3-7, optionally setting the reaction time in the reaction tank to 20 minutes;

[0130] 26. Perform signal acquisition, optionally including image acquisition and grayscale analysis.

[0131] To demonstrate the reliability of the detection chip, a full-process simulation was performed using both dye solutions and real reagents, as shown below. Figure 9 and Figure 10 As shown in the figure, the results demonstrate that the designed chip principle can be practically implemented and has high compatibility with different detection reagents, going beyond mere theory.

[0132] In summary, the microfluidic detection chip of this invention enables fully automated biochemical reactions. Through a separate centrifuge device and the chip, it allows for the selective and sequential release of corresponding reagent chambers, high-throughput quantitative distribution and metered collection of reagents. Simultaneously, it can automate complex and repetitive reaction processes, achieving high-throughput and precise reactions. Furthermore, the entire complex process is completed by the chip and a simple centrifuge device, eliminating the need for professional testing personnel. Combined with the miniaturized characteristics of microfluidics, it saves reagent consumption, significantly accelerates detection speed, reduces detection costs, and eliminates the impact of sample aerosols on specimens and the risk of infection to testing personnel. Therefore, the microfluidic detection chip provided by this invention has broad application value in fields including nucleic acid, immunology, biochemistry, and ion analysis.

[0133] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A microfluidic detection chip having a rotation center, characterized in that, The application relates to a microfluidic chip for detecting a plurality of samples, comprising one or more detection units; wherein the detection unit comprises: a spiral-shaped dosing microchannel arranged around the rotation center, one or more first liquid pools arranged in the inside of the dosing microchannel, a plurality of reaction units arranged along the dosing microchannel, and a first waste liquid pool connected with the end of the dosing microchannel; wherein each first liquid pool is connected with the head end of the dosing microchannel through a first siphon valve, and a sealable sample adding hole is arranged on the first siphon valve; the bending part at the beginning of the first siphon valve is hydrophilic; the reaction unit comprises: a second liquid pool arranged in the inside of the dosing microchannel, a metering pool, a reaction pool and a second waste liquid pool arranged in sequence from the inside to the outside of the dosing microchannel; the second liquid pool is connected with the metering pool through a first microchannel, the metering pool is connected with the reaction pool through a second siphon valve, the reaction pool is connected with the second waste liquid pool through a second microchannel, and the second waste liquid pool is connected with the second siphon valve through a gas path balance microchannel; the reaction pool is connected with the outside through a gas hole, the volume of the reaction pool is greater than that of the metering pool, an independent sample adding hole is arranged on the second liquid pool, and the bending part at the beginning of the second siphon valve is hydrophilic; the connection point of the gas path balance microchannel and the second siphon valve is the connection point of the second siphon valve and the reaction pool, or the connection point of the gas path balance microchannel and the second siphon valve is the end point of the hydrophilic treated part of the second siphon valve, or the connection point of the gas path balance microchannel and the second siphon valve is located between the connection point of the second siphon valve and the reaction pool and the end point of the hydrophilic treated part of the second siphon valve. The connection point of one of the first liquid pools and the first siphon valve is higher than the bottom end of the first siphon valve. The reaction pool is an oval-shaped chamber with two micro-protrusions, and the two micro-protrusions are respectively the connection points of the second siphon valve and the gas hole and the reaction pool. The metering pool is a square combined with a bottom triangular chamber. The second microchannel is a meandering microchannel. The first liquid pool is a fan ring-shaped chamber. The connection point of the gas path balance microchannel and the second siphon valve is the end point of the hydrophilic treated part of the second siphon valve.

2. The microfluidic detection chip of claim 1, wherein, The microfluidic chip is a multi-layer structure, and comprises, from bottom to top, a bottom cover, a siphon valve layer, a microchannel layer, a chamber layer and an upper cover.

3. The microfluidic detection chip of claim 1 or 2, wherein, The first siphon valve and the second siphon valve are located in the siphon valve layer.

4. The microfluidic detection chip of claim 1 or 2, wherein, The dosing microchannel, the first microchannel, the second microchannel and the gas path balance microchannel are located in the microchannel layer.

5. The microfluidic detection chip of claim 1 or 2, wherein, The first liquid pool, the first waste liquid pool, the second liquid pool, the metering pool, the reaction pool and the second waste liquid pool are located in the chamber layer.

6. The microfluidic detection chip of claim 1 or 2, wherein, The gas hole, the sealable sample adding hole and the independent sample adding hole are located in the upper cover.

7. The microfluidic detection chip of claim 1 or 2, wherein, ​ 8. The microfluidic detection chip of claim 1 or 2, wherein, ​ ​ ​ ​ ​

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