Microfluidic chip and method for detecting allergens

Abstract

The present invention relates to a microfluidic chip and method for detecting allergens. The microfluidic chip comprises a chip body and a liquid capsule assembly, wherein the chip body is provided with a sample injection chamber, a whole blood separation structure, a mixing chamber, an arc-shaped liquid separation flow channel, a plurality of reaction units and a substrate reagent chamber; the whole blood separation structure is connected to the sample injection chamber and the mixing chamber; the liquid separation flow channel is connected to the mixing chamber; a plurality of reaction units are distributed at intervals along the rotation circumferential direction, each reaction unit comprises an immune reaction chamber and a reaction waste liquid chamber connected to the immune reaction chamber, the immune reaction chamber is pre-loaded with allergen freeze-dried beads and labeled antibody freeze-dried beads, and each immune reaction chamber is connected to the liquid separation flow channel; the liquid capsule assembly comprises a first chamber, a second chamber, a third chamber, a first liquid capsule storing a plasma diluent, a second liquid capsule storing a cleaning solution and a third liquid capsule storing a luminescent substrate diluent, the first liquid capsule is located in the first chamber, the second liquid capsule is located in the second chamber, and the third liquid capsule is located in the third chamber.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay detection, and in particular to a microfluidic chip and method for detecting allergens. Background Art

[0002] Hypersensitivity is an immune response characterized by tissue damage or functional disorder after the body is continuously stimulated by an antigen or stimulated by the same antigen again. According to the mechanism and clinical characteristics of hypersensitivity, it is divided into types I, II, III and IV. Type I hypersensitivity refers to a reaction mediated by immunoglobulin E (IgE), involving mast cells in tissues and basophils in peripheral blood, which causes local or systemic physiological disorders and tissue damage by releasing bioactive mediators. Diseases caused by type I hypersensitivity mainly include: allergic conjunctivitis of the eyes; allergic rhinitis of the nose; allergic asthma and allergic bronchopulmonary aspergillosis of the trachea and lungs; allergic gastroenteritis of the digestive tract; atopic dermatitis, allergic urticaria, allergic angioedema, allergic immediate contact reactions of the skin; and allergic diseases with severe systemic reactions.

[0003] Substances that cause type I hypersensitivity reactions are called allergens, also known as allergens. Allergens that are exposed through the respiratory tract are called inhaled allergens, which mainly include plant pollen, animal dander, fungal spores or hyphae, dust mites, etc. Allergens ingested through the digestive tract are called food allergens, mainly including milk, eggs, fish, shrimp, crab, nuts, etc. The key to preventing and treating allergic diseases is to discover allergens and avoid contact with them. The diagnosis of allergic diseases includes asking about medical history, in vitro diagnosis and in vivo testing, among which in vitro diagnosis plays an important role, and allergen testing is one of the important in vitro diagnostic items.

[0004] Allergen testing does not actually detect allergen antigens, but indirectly infers whether the person being tested is allergic to the corresponding substance by detecting allergen-specific IgE (sIgE) antibodies. As the etiological diagnosis basis for allergic diseases, allergen sIgE antibody testing is of great value in diagnosing allergic patients, guiding desensitization treatment, evaluating the effect of specific desensitization treatment, and assessing the risks of contact with allergens. At the same time, screening allergens is also very important for the prevention of suspected allergic patients. At present, the serological detection methods of allergen sIgE antibodies mainly include dot enzyme-linked immunosorbent assay (Dot-ELISA), ELISA, and fluorescence enzyme immunoassay (FEIA).

[0005] With the advancement of technology, microfluidic chip technology (Microfluidics) can easily realize the automation of biological, chemical, and medical analysis processes. Due to its huge potential in the fields of biology, chemistry, medicine, etc., it has developed into a popular research direction and has been applied to the field of in vitro diagnosis. However, the current microfluidic chips based on microfluidic technology cannot solve the problem of joint detection of multiple allergens; and the microfluidic chips for blood samples still need to process samples in advance, and individual steps in the detection process still need to be completed by human cooperation, and the overall degree of automation is still relatively low; in addition, the current microfluidic chips have more stringent requirements for storage and transportation conditions, and generally need to be stored in an environment of 2 to 8°C and transported in a cold chain. If these conditions are not met, it is easy to cause uncontrollable effects on the analytical performance of the reagents, and even cause the reagents to fail. Summary of the invention

[0006] Based on this, it is necessary to provide a microfluidic chip that can detect multiple allergens simultaneously, has a high degree of automation, and is easy to transport and store.

[0007] A microfluidic chip for detecting allergens, comprising:

[0008] A chip body, the chip body having a rotation center, and the chip body is provided with:

[0009] Injection cavity;

[0010] A whole blood separation structure, comprising a plasma chamber, a blood cell chamber and a blood waste liquid chamber, wherein the plasma chamber is connected to the sample injection chamber, the blood cell chamber and the blood waste liquid chamber are respectively connected to the plasma chamber, and the blood cell chamber is further away from the rotation center than the plasma chamber;

[0011] A mixing chamber, connected to the plasma chamber;

[0012] An arc-shaped liquid separation channel, the liquid separation channel comprises a main channel and a plurality of cup-separating cavities, the main channel has an inlet end and an outlet end, the inlet end is connected to the mixing cavity; the main channel is arc-shaped, the plurality of cup-separating cavities are arranged at intervals in the circumferential direction of the main channel and are connected to the main channel, and the distance from the main channel to the rotation center gradually increases from the inlet end to the outlet end;

[0013] A plurality of reaction units, wherein the plurality of reaction units are spaced apart along the circumferential direction of rotation, wherein the reaction units include an immune reaction chamber and a reaction waste liquid chamber connected to the immune reaction chamber, wherein the immune reaction chamber is correspondingly connected to the cup chamber, and the cup chamber, the immune reaction chamber and the reaction waste liquid chamber are sequentially arranged along the radial direction of the main channel away from the rotation center; allergen freeze-dried beads and labeled antibody freeze-dried beads are pre-loaded in some of the immune reaction chambers, wherein the allergen freeze-dried beads include allergens and carriers, and the allergens in each immune reaction chamber are different;

[0014] A substrate reagent chamber pre-loaded with luminescent substrate freeze-dried beads, the substrate reagent chamber being connected to the mixing chamber;

[0015] A liquid capsule component, the liquid capsule component includes a first liquid capsule storing a plasma diluent, a second liquid capsule storing a cleaning liquid, and a third liquid capsule storing a luminescent substrate diluent, the plasma diluent in the first liquid capsule can enter the mixing chamber, the cleaning liquid in the second liquid capsule can enter the mixing chamber, and the luminescent substrate diluent in the third liquid capsule can enter the mixing chamber through the substrate reagent chamber.

[0016] The above-mentioned microfluidic chip for detecting allergens is provided with a sample injection chamber, a whole blood separation structure, a mixing chamber, a liquid separation channel, a plurality of reaction units and a substrate reagent chamber on the chip body, and is matched with a liquid capsule component, so that the above-mentioned microfluidic chip can test multiple allergens by adding samples once, and there is no need to pre-treat the whole blood sample separately, and no human cooperation is required during the detection process. All reagents are integrated into the microfluidic chip, and the complete detection process can be completed without the help of a liquid addition system, universal reagents and cleaning fluid outside the chip. The operation is simple, time-saving and highly automated; and because multiple reaction units share the sample injection chamber, the whole blood separation structure, the mixing chamber and the liquid separation channel, this saves space and enables the above-mentioned microfluidic chip to arrange more than 12 reaction units in a limited area, so that more than 12 allergens can be detected simultaneously, thereby making the detection of allergens integrated; in addition, the allergens, the labeled antibodies and the luminescent substrates are in the form of freeze-dried beads, which enables the above-mentioned microfluidic chip to be stored at room temperature and is also convenient for transportation.

[0017] In one embodiment, there are at least 12 reaction units; each of the allergens is independently derived from the following group: house dust mites; dust mites; house dust; cat hair; dog hair; cockroaches; mold; pollen; humulum; mugwort; ragweed; egg white; egg yolk; milk; peanuts; soybeans; beef; mutton; shrimp; crab; seafood; allergenic fruits; nuts; CCD.

[0018] In one embodiment, the allergenic fruit includes at least one of peach, apple, mango, lychee and strawberry; and / or the pollen includes at least one of willow pollen, poplar pollen and elm pollen; and / or the seafood includes at least one of cod, lobster and scallop; and / or the nuts include at least one of cashew, pistachio, hazelnut, almond and walnut; and / or the mold includes at least one of Penicillium, Aspergillus fumigatus, Alternaria alternata and Cladosporium.

[0019] In one embodiment, some of the immune reaction chambers are pre-loaded with background quality control freeze-dried beads, and some of the immune reaction chambers are pre-loaded with reagent degradation quality control freeze-dried beads.

[0020] In one embodiment, the carrier and the allergen can be coupled via avidin and biotin.

[0021] In one embodiment, the carrier is a magnetic bead connected with streptavidin, and the allergen is coupled with biotin.

[0022] In one embodiment, the allergen freeze-dried beads, the labeled antibody freeze-dried beads and the luminescent substrate freeze-dried beads each independently contain a freeze-dried diluent after freeze-drying, and the freeze-dried diluent before freeze-drying includes a buffer, an auxiliary agent with a weight percentage of 3% to 20%, a surfactant with a weight percentage of 0.01% to 1% and a preservative with a weight percentage of 0.01% to 0.5%; the auxiliary agent includes at least one of mannitol, trehalose, sucrose, lactose, dextran, PEG, PVP, BSA and gelatin.

[0023] In one embodiment, the surfactant includes at least one of Tween 20, Tween 80, Span 80, Triton X-45, and Triton X-100;

[0024] And / or, the buffer is TBS buffer;

[0025] Furthermore, the allergen freeze-dried beads also contain at least one of a protein protectant and an antioxidant; and / or the labeled antibody freeze-dried beads contain at least one of a protein protectant and an antioxidant.

[0026] In some embodiments, the liquid capsule assembly has a first cavity, a second cavity and a third cavity, the first cavity and the second cavity are respectively connected to the mixing cavity, the third cavity is connected to the mixing cavity via the substrate reagent cavity, the first liquid capsule is located in the first cavity, the second liquid capsule is located in the second cavity, and the third liquid capsule is located in the third cavity.

[0027] A method for detecting allergens, using the above-mentioned microfluidic chip for detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work, among which:

[0029] Figure 1 The first is a microfluidic chip of an embodiment;

[0030] Figure 2 for Figure 1 An exploded view of the microfluidic chip is shown;

[0031] Figure 3 for Figure 1 A three-dimensional diagram of a chip body of the microfluidic chip shown;

[0032] Figure 4 for Figure 1 A top view of the chip body of the microfluidic chip is shown.

[0033] Reference numerals:

[0034] 10. Microfluidic chip; 110. Chip body; 110a. Covering surface; 111. Sample injection chamber; 112. Whole blood separation structure; 112a. Plasma chamber; 112b. Blood cell chamber; 112c. Blood waste liquid chamber; 112d. Platform part; 112e. Collection part; 112f. Blood sample sufficient volume detection chamber; 113. Mixing chamber; 114. Liquid separation channel; 114a. Main flow channel; 114b. Cup separation chamber; 114c. Waste liquid separation chamber; 114d, sacrificial chamber; 115, reaction unit; 115a, immune reaction chamber; 115b, reaction waste liquid chamber; 115c, first microfluidic channel; 115d, second microfluidic channel; 116, substrate reagent chamber; 117, sedimentation tank; 118, first siphon channel; 119, second siphon channel; 120, liquid capsule assembly; 121, first liquid capsule; 122, second liquid capsule; 123, third liquid capsule; 130, cover plate; 140, adhesive layer. DETAILED DESCRIPTION

[0035] In order to facilitate the understanding of the present invention, the present invention will be described more fully below, and the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0036] It should be noted that when an element is described as being "fixed to" another element, it can be directly on the other element, or one or more intermediate elements can exist between them. When an element is described as being "connected to" another element, it can be directly connected to the other element, or one or more intermediate elements can exist between them.

[0037] When the terms "vertical", "horizontal", "left", "right", "up", "down", "inside", "outside", "bottom" and the like are used to indicate an orientation or positional relationship, it is based on the orientation or positional relationship shown in the drawings and is only for the convenience of description. It does not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore cannot be understood as a limitation on the present application. In addition, the terms "first", "second", etc. are used for descriptive purposes only and cannot be understood as indicating or implying relative importance. The term "and / or" includes any and all combinations of one or more related listed items. Herein, "optionally" means by way of example. The term "multiple" means at least two; the term "multiple" means at least two.

[0038] It should be noted that, in this article, the capacity of the chamber refers to the maximum amount of liquid that the chamber can hold; the depth of the components on the chip body refers to the distance from the bottom of the corresponding component to the cover surface, for example, the depth of the collection part refers to the distance from the bottom of the collection part to the cover surface. In addition, in this article, unless otherwise specified, the bottom surface of each component is a flat surface, that is, the distance from each position on the bottom surface of the component to the cover surface is equal.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0040] See also Figure 1 and Figure 2In one embodiment of the present application, a microfluidic chip 10 for detecting allergens is provided. The microfluidic chip 10 includes a chip body 110 and a liquid capsule component 120. The chip body 110 has a rotation center. The chip body 110 is provided with a sampling chamber 111, a whole blood separation structure 112, a mixing chamber 113, an arc-shaped liquid separation channel 114, a plurality of reaction units 115 and a substrate reagent chamber 116. The whole blood separation structure 112 has a plasma chamber 112a, a blood cell chamber 112b and a blood waste liquid chamber 112c. The plasma chamber 112a is connected to the sampling chamber 111, and the plasma The chamber 112a is connected to the blood cell chamber 112b, the blood waste liquid chamber 112c is connected to the plasma chamber 112a, and the blood cell chamber 112b is farther away from the rotation center than the plasma chamber 112a; the mixing chamber 113 is connected to the plasma chamber 112a; the liquid separation channel 114 includes a main channel 114a and a plurality of cup chambers 114b, the main channel 114a has an inlet end and an outlet end, and the inlet end is connected to the mixing chamber 113; the main channel 114a is arc-shaped, and the plurality of cup chambers 114b are arranged at intervals in the circumferential direction of the main channel 114a and are connected to the main channel 114a, and the main channel 114a to the rotation center is connected to the main channel 114a. The distance from the rotation center increases gradually from the inlet end to the outlet end; multiple reaction units 115 are spaced apart along the rotation circumference, and each reaction unit 115 includes an immune reaction chamber 115a and a reaction waste liquid chamber 115b connected to the immune reaction chamber 115a. The immune reaction chamber 115a is correspondingly connected to the cup chamber 114b. The cup chamber 114b, the immune reaction chamber 115a and the reaction waste liquid chamber 115b are sequentially arranged in the radial direction of the main channel 114a away from the rotation center. A part of the immune reaction chamber 115a is pre-loaded with allergen freeze-dried beads and labeled antibody freeze-dried beads. The allergens in the immune reaction chamber 115a are different; the substrate reagent chamber 116 pre-loaded with luminescent substrate freeze-dried beads is connected to the mixing chamber 113; the liquid capsule assembly 120 includes a first liquid capsule 121 storing a plasma diluent, a second liquid capsule 122 storing a cleaning liquid, and a third liquid capsule 123 storing a luminescent substrate diluent. The plasma diluent in the first liquid capsule 121 can enter the mixing chamber 113, the cleaning liquid in the second liquid capsule 122 can enter the mixing chamber 113, and the luminescent substrate diluent in the third liquid capsule 123 can enter the mixing chamber 113 through the substrate reagent chamber 116.

[0041] The microfluidic chip 10 is provided with a sampling chamber 111, a whole blood separation structure 112, a mixing chamber 113, a liquid separation channel 114, a plurality of reaction units 115 and a substrate reagent chamber 116 on the chip body 110, and is equipped with a liquid capsule component 120. During detection, the blood sample to be tested enters the chip body 110 through the sampling chamber 111, enters the mixing chamber 113 after the serum or plasma is separated by the whole blood separation mechanism, and then enters the plurality of reaction units 115 through the flow channel after being mixed with the plasma diluent. When the blood sample to be tested contains sIgE, sIgE forms a complex with the allergen and the labeled antibody in the immune reaction chamber 115a, reacts with the luminescent substrate after being cleaned by the cleaning solution, and the amount of sIgE in the blood sample to be tested (qualitative or quantitative) can be determined by detecting the luminescent signal. Therefore, the microfluidic chip 10 can test multiple allergens by adding samples once, and does not need to pre-treat the whole blood sample separately. The detection process does not require human cooperation. All reagents are integrated into the microfluidic chip. The complete detection process can be completed without the help of the liquid adding system, general reagents, cleaning fluid and other consumables outside the chip. The operation is simple, time-saving and highly automated. In addition, since multiple reaction units 115 share the injection chamber 111, the whole blood separation structure 112, the mixing chamber 113 and the liquid separation channel 114, and the structural design of the liquid separation channel 114, this saves space and enables the microfluidic chip 10 to arrange more than 12 reaction units 115 in a limited area, so that more than 12 allergens can be detected at the same time, thereby making the detection of allergens integrated; in addition, allergens, labeled antibodies and luminescent substrates are all in the form of freeze-dried beads, which enables the microfluidic chip 10 to be stored at room temperature and is also convenient for transportation. In addition, since the plasma diluent, cleaning solution and luminescent substrate diluent are all dispensed into the liquid capsule assembly 120 and integrated on the microfluidic chip 10, there is no need to separately equip external general reagents and consumables, and the supporting detection equipment and instruments do not need to be equipped with liquid path mechanisms, nor do they need to perform liquid path maintenance, so the maintenance cost of the supporting detection equipment is lower.

[0042] Optionally, the material of the chip body 110 includes, but is not limited to, glass, ABS, PDMS, PMMA, PP, PET, or PC. In some embodiments, the material of the chip body 110 is an opaque material. Opaque materials as the material of the chip body 110 are more conducive to subsequent detection. Optionally, the material of the chip body 110 is a colored opaque material. In some embodiments, the diameter of the chip body 110 is 100 mm to 140 mm. Optionally, the diameter of the chip body 110 is 100 mm, 120 mm, 130 mm, or 140 mm.

[0043] See also Figure 3 and Figure 4Specifically, the injection chamber 111 is used to hold the added sample. In the present embodiment, the capacity of the injection chamber 111 is above 400 μL. According to the above arrangement, the amount of at least 20 reaction units 115 can be met. Further, the capacity of the injection chamber 111 is above 450 μL. Furthermore, the capacity of the injection chamber 111 is 400 μL to 600 μL. Optionally, the injection chamber 111 is fan-shaped, and the injection chamber 111 protrudes from the rotation center toward the edge of the chip body 110. In the illustrated embodiment, the width of the injection chamber 111 gradually increases in a clockwise direction. According to such an arrangement, it is convenient for the sample to enter the downstream through centrifugation, reduce the centrifugation time, and improve the detection efficiency.

[0044] Specifically, the whole blood separation structure 112 is used to process the blood sample to separate serum or plasma from the whole blood for downstream detection. The whole blood separation structure 112 includes a plasma chamber 112a, a blood cell chamber 112b and a blood waste liquid chamber 112c. During centrifugation, the whole blood is separated into serum or plasma and blood cells, and the serum or plasma stays in the plasma chamber 112a while the blood cells stay in the blood cell chamber 112b, thereby achieving separation. When the blood in the plasma chamber 112a exceeds its capacity, it enters the blood waste liquid chamber 112c, thereby achieving quantification of the blood. Further, the chip body 110 has a cover surface 110a; the plasma chamber 112a has a flow-aiding surface, and the distance from the flow-aiding surface to the cover surface 110a gradually decreases along the direction from the entrance of the plasma chamber 112a to the outlet of the plasma chamber 112a. Through the setting of the flow-aiding surface, the blood cells in the plasma chamber 112a are more likely to enter the blood cell chamber 112b during centrifugation, so that the whole blood separation is more thorough, which is conducive to subsequent detection. Optionally, the plasma chamber 112a is generally funnel-shaped, and the distance between the side walls of the plasma chamber 112a gradually narrows in the reverse direction from the entrance of the plasma chamber 112a to the exit of the plasma chamber 112a. It is understood that the shape of the plasma chamber 112a is not limited to the above, and the shape of the blood cell chamber 112b is also not limited.

[0045] In this embodiment, the volume ratio of the plasma chamber 112a to the blood cell chamber 112b is 1:(1-5). The volume ratio of the plasma chamber 112a to the blood cell chamber 112b is set to 1:(1-5) to fully separate the plasma from the blood cells, so that there is no other interference such as hemoglobin in the plasma. Furthermore, the volume ratio of the plasma chamber 112a to the blood cell chamber 112b is 1:(2-5). Setting the volume ratio of the plasma chamber 112a to the blood cell chamber 112b to 1:(2-5) can make the whole blood separation structure 112 compatible with normal whole blood samples of almost all age groups and genders. In an optional specific example, the volume ratio of the plasma chamber 112a to the blood cell chamber 112b is 1:2, 1:3, 1:4 or 1:5.

[0046] Specifically, the blood waste liquid chamber 112c is used to hold whole blood that exceeds the carrying capacity of the plasma chamber 112a, that is, to hold excess whole blood. During use, excess whole blood will overflow and enter the blood waste liquid chamber 112c. In the illustrated embodiment, the entrance of the blood waste liquid chamber 112c is close to the entrance of the plasma chamber 112a. It is understandable that in other embodiments, the entrance of the plasma chamber 112a is not limited to this, and can also be other positions, as long as it can receive the whole blood overflowing from the plasma chamber 112a. Further, the blood waste liquid chamber 112c includes a platform portion 112d and a collecting portion 112e, the platform portion 112d is connected to the plasma chamber 112a, and the collecting portion 112e is connected to the platform portion 112d, and the depth of the platform portion 112d is less than the depth of the collecting portion 112e. When in use, the whole blood overflowing from the plasma chamber 112a flows into the collecting portion 112e through the platform portion 112d. By designing the platform portion 112d and the collecting portion 112e, the whole blood entering the blood waste liquid chamber 112c is not easy to flow back to the plasma chamber 112a. Furthermore, the chip body 110 also has a blood sample sufficient amount detection chamber 112f for reflecting whether the amount of whole blood is sufficient for subsequent detection. The blood sample sufficient amount detection chamber 112f is connected to the blood waste liquid chamber 112c and is farther away from the rotation center than the blood waste liquid chamber 112c. In the illustrated embodiment, the blood sample sufficient amount detection chamber 112f is connected to the platform portion 112d, and the blood sample sufficient amount detection chamber 112f and the collecting portion 112e are spaced apart and located at one end of the platform portion 112d away from the rotation center, and the blood sample sufficient amount detection chamber 112f is closer to the blood cell chamber 112b.

[0047] In some embodiments, the waste blood chamber 112c is further provided with a vent hole, which facilitates the waste blood to enter the waste blood chamber 112c.

[0048] In some embodiments, the chip body 110 is further provided with a sedimentation tank 117. Specifically, the sedimentation tank 117 is used to settle a portion of naturally occurring agglomerates in the whole blood sample and fibers or particles introduced during sample addition. For example, possible clots in the blood sample, including blood clots, larger fat masses, etc., are prevented from clotting the flow channel in the chip. The sedimentation tank 117 is located between the injection chamber 111 and the plasma chamber 112a, and the injection chamber 111 and the plasma chamber 112a are connected through the sedimentation tank 117.

[0049] Specifically, the mixing chamber 113 is a place where serum or plasma is evenly mixed with the diluent, and is also the only way for the cleaning liquid to enter the liquid separation channel 114, and is also a place where the luminescent substrate and the luminescent substrate diluent are evenly mixed. The mixing chamber 113 is connected to the plasma chamber 112a. More specifically, the mixing chamber 113 is connected to the plasma chamber 112a through the first siphon channel 118. In this embodiment, the width of the first siphon channel 118 is 0.2mm~1.5mm, and the depth of the first siphon channel 118 is 0.1mm~1mm. The size of the first siphon channel 118 is arranged according to the above and linked to other associated structures so that the accurately quantified plasma can be completely drained from the plasma chamber 112a to the downstream mixing chamber 113 after the whole blood separation step is completed, and the blood cells in the blood cell chamber 112b during the whole blood separation step will not be drained into the mixing chamber 113, ensuring that the whole blood does not pass through and the separated plasma passes, so that the blood cells will not interfere with the accuracy of the subsequent reagent test results. In the illustrated embodiment, the mixing chamber 113 is substantially crescent-shaped around the rotation center. It is understood that in other embodiments, the shape of the mixing chamber 113 is not limited to the above, and may also be other shapes.

[0050] In some embodiments, one-third of the area of ​​the first siphon flow channel 118 near the mixing chamber 113 is coated with a hydrophobic reagent, and two-thirds of the area of ​​the first siphon flow channel 118 near the main channel 114a is coated with a hydrophilic reagent. The above-mentioned coating treatment can effectively enhance the siphon effect of the first siphon flow channel 118. The coating method can be selected from ultrasonic spraying, micro-spotting, etc. In an optional specific example, an ultrasonic spraying scheme is selected. The position that does not need to be sprayed is made into a mask, covered on the chip body 110, and placed in an ultrasonic spraying instrument. The instrument sprays the reagent in the form of particles to the chip surface not covered by the mask, and the reagent adheres to the surface of the microchannel. In an optional specific example, the width of the first siphon flow channel 118 is 0.1-1.0 mm, and the depth of the first siphon flow channel 118 is 0.1-1.0 mm.

[0051] Specifically, the arc-shaped liquid separation channel 114 is used to quantitatively transport the liquid in the mixing chamber 113 to the reaction unit 115. The liquid separation channel 114 includes a main channel 114a and a plurality of spaced-apart cup chambers 114b. When in use, as the centrifuge is performed, the liquid flowing out of the mixing chamber 113 enters the main channel 114a along the centrifugal direction and gradually fills each cup chamber 114b. The main channel 114a is arc-shaped, and the main channel 114a protrudes from the rotation center to the edge of the chip body 110, and the plurality of cup chambers 114b are distributed at intervals along the circumference of the main channel 114a on the side away from the rotation center of the main channel 114a. In this embodiment, the width of the main channel 114a is 0.5mm to 3mm, and the depth of the main channel 114a is 1.5mm to 3mm. In an optional specific example, the width of the main channel 114a is 1mm to 2mm, and the depth of the main channel 114a is 0.5mm to 3.5mm.

[0052] In some embodiments, the main flow channel 114a is in an outer spiral shape with the rotation center as the center, and a plurality of cup-dividing chambers 114b are arranged in parallel at equal angles and are directly connected to the main flow channel 114a on the outer periphery of the main flow channel 114a with the rotation center as the center. In the direction from the inlet end to the outlet end of the main flow channel 114a, the ratio of the distance from the first cup-dividing chamber 114b to the rotation center to the distance from the last cup-dividing chamber 114b to the rotation center is 1:(1.05-1.2). According to such an arrangement, when there are more cup-dividing chambers 114b (for example, the number of cup-dividing chambers 114b is more than 25), each cup-dividing chamber 114b can be filled with liquid from the mixing chamber 113 at a lower rotation speed. In the illustrated embodiment, the ratio of the distance from the first cup-dividing chamber 114b to the rotation center to the distance from the last cup-dividing chamber 114b to the rotation center is 1:1.12.

[0053] In some embodiments, with the rotation center as the vertex, the angle formed by the rotation center and the inlet end and the outlet end is 0° to 359°. It is understandable that the angle formed by the rotation center and the inlet end and the outlet end is not limited to 0° to 359°, and if the disk size of the chip body 110 is appropriate, it can also exceed 359°, such as 400°, 480°, etc.

[0054] In this embodiment, the mixing chamber 113 is connected to the liquid separation channel 114 through the second siphon channel 119, the width of the second siphon channel 119 is 0.2mm~1.5mm, and the depth of the second siphon channel 119 is 0.1mm~1mm. The size of the second siphon channel 119 is set as above so that the fully diluted sample can be fully and evenly drained into the main channel 114a. In an optional specific example, the width of the second siphon channel 119 is 0.5mm, and the depth of the second siphon channel 119 is 0.3mm. In the illustrated embodiment, the first siphon channel 118 and the second siphon channel 119 are both located at one end of the mixing chamber 113 close to the plasma chamber 112a.

[0055] In some embodiments, the second siphon flow channel 119 is coated with a hydrophilic reagent in the portion near the mixing chamber 113, the middle portion of the second siphon flow channel 119 is coated with a hydrophobic reagent, and the portion of the second siphon flow channel 119 near the liquid separation flow channel 114 is coated with a hydrophilic reagent. The coating treatment can effectively enhance the siphon effect of the second siphon flow channel 119. The coating method can be ultrasonic spraying, micro-spotting, etc. In an optional specific example, an ultrasonic spraying scheme is selected. The position where no spraying is required is made into a mask, covered on the chip body 110, and placed in an ultrasonic spraying instrument. The instrument sprays the reagent in the form of particles to the chip surface not covered by the mask, and the reagent adheres to the surface of the microchannel. In an optional specific example, the width of the second siphon flow channel 119 is 0.1mm~1.0mm, and the depth of the second siphon flow channel 119 is 0.1mm~1.0mm.

[0056] In the illustrated embodiment, the liquid separation channel 114 further includes a waste liquid separation chamber 114c located at the liquid outlet end of the main channel 114a. The waste liquid separation chamber 114c is used to contain excess liquid flowing out of the main channel 114a.

[0057] In the illustrated embodiment, the liquid separation channel 114 further includes a sacrificial chamber 114d. The sacrificial chamber 114d is connected to the main channel 114a and is separated from the cup-separating chamber 114b. The sacrificial chamber 114d is closer to the inlet end than the cup-separating chamber 114b. The sacrificial chamber 114d is arranged so that the first cup-separating chamber 114b counted from the inlet end is not likely to have the actual liquid filled less than the preset volume, thereby affecting the next reaction; at the same time, the sacrificial chamber 114d can also effectively reduce the liquid impact force borne by the first cup-separating chamber 114b, thereby improving the accuracy of the first immune reaction chamber 115a.

[0058] Specifically, a plurality of reaction units 115 are spaced apart along the circumferential direction of rotation, and the reaction unit 115 includes an immune reaction chamber 115a and a reaction waste liquid chamber 115b connected to the immune reaction chamber 115a, and each immune reaction chamber 115a is connected to the liquid separation channel 114. The cup separation chamber 114b, the immune reaction chamber 115a and the reaction waste liquid chamber 115b are sequentially arranged in the radial direction of the main channel 114a in a direction away from the rotation center.

[0059] Furthermore, the cup chamber 114b is connected to the immune reaction chamber 115a through the first microchannel 115c, and the immune reaction chamber 115a is connected to the reaction waste liquid chamber 115b through the second microchannel 115d. The first microchannel 115c is used to prevent the liquid from entering the immune reaction chamber 115a during the process of the liquid gradually entering the cup chamber 114b, and to guide the liquid in the cup chamber 114b to enter the immune reaction chamber 115a after the liquid fills all the cup chambers 114b. The second microchannel 115d is used to prevent the substances in the immune reaction chamber 115a from entering the reaction waste liquid chamber 115b during the immune reaction process, and to guide the liquid in the immune reaction chamber 115a to enter the reaction waste liquid chamber 115b when it needs to be discarded.

[0060] In this embodiment, the width of the first microchannel 115c and the second microchannel 115d are independently 0.2mm to 0.7mm, and the depth of the first microchannel 115c and the second microchannel 115d are independently 0.02mm to 0.07mm; the length of the first microchannel 115c is 1.5mm to 2.5mm, and the length of the second microchannel 115d is 3.5mm to 5.5mm. According to the above arrangement, it is more conducive to allow the liquid in the scoring cup chamber 114b to enter the immune reaction chamber 115a at a lower speed without flowing out from the second microchannel 115d. When the liquid in the immune reaction chamber 115a needs to be centrifuged into the reaction waste liquid chamber 115b, the liquid can also smoothly enter the reaction waste liquid chamber 115b. In some embodiments, the first microchannel 115c and the second microchannel 115d are both microchannels that have been hydrophobically treated. Furthermore, the first microchannel 115c and the second microchannel 115d are coated with a hydrophobic reagent, respectively, which can effectively increase the obstruction effect, so that multiple cleanings can be completed smoothly. The coating method can be selected from ultrasonic spraying, micro-spotting, etc. In an optional specific example, an ultrasonic spraying scheme is selected. The position that does not need to be sprayed is made into a mask, covered on the chip body 110, and placed in an ultrasonic spraying instrument. The instrument sprays the reagent in the form of particles to the surface of the chip not covered by the mask, and the reagent adheres to the surface of the microchannel. In this embodiment, after the hydrophobic treatment of the first microchannel 115c and the second microchannel 115d, it is more conducive to the speed difference to achieve the obstruction and liquid guiding effect of the first microchannel 115c and the second microchannel 115d, so as to meet the multiple cleaning and drainage processes of the chip.

[0061] In some embodiments, the distance between the bottom of the immune reaction chamber 115 a and the lower surface of the chip body 110 is not less than 0.3 mm.

[0062] In the illustrated embodiment, the immune reaction chamber 115a is cylindrical, and the positive projection of the reaction waste liquid chamber 115b on the cover surface 110a is an isosceles trapezoid. It is understandable that in other embodiments, the shapes of the immune reaction chamber 115a and the reaction waste liquid chamber 115b are not limited to the above, and can also be other shapes.

[0063] In some embodiments, a portion of the immune reaction chamber 115a is pre-loaded with labeled antibodies and allergen freeze-dried beads, and another portion of the immune reaction chamber 115a is pre-loaded with quality control freeze-dried beads. It is understood that the marker of the labeled antibody is not particularly limited. The allergen freeze-dried beads include an allergen and a carrier. Optionally, the allergens in each immune reaction chamber 115a are independently derived from the following group: house dust mites; dust mites; house dust; cat hair; dog hair; cockroaches; mold; pollen; humulus; mugwort; ragweed; egg white; egg yolk; milk; peanuts; soybeans; beef; mutton; shrimp; crab; seafood; allergenic fruits; nuts; CCD (cross-reactive carbohydrate determinant clusters). Further, the mold includes at least one of Penicillium, Aspergillus fumigatus, Alternaria and Cladosporium; and / or, the pollen includes at least one of willow pollen, poplar pollen and elm pollen; and / or, the seafood includes at least one of cod, lobster and scallop; and / or, the allergenic fruit includes at least one of peach, apple, mango, lychee and strawberry; and / or, the nuts include at least one of cashew, pistachio, hazelnut, almond and walnut. It is understandable that the allergens are not limited to the above, but can also be other substances.

[0064] Optionally, the allergen and the carrier exist in the form of freeze-dried beads that are independent of each other before use. Of course, the allergen and the carrier may have been connected before use. Optionally, the carrier and the allergen can be coupled through avidin and biotin. In this embodiment, the carrier is a magnetic bead connected with streptavidin, and biotin is coupled to the allergen. Through the action of biotin and streptavidin, the allergen is connected to the carrier. It is understandable that in other embodiments, the connection method between the allergen and the carrier is not limited to the merchant's avidin and biotin, and can also be other connection methods.

[0065] Optionally, the quality control freeze-dried beads include at least one of background quality control freeze-dried beads and reagent degradation quality control freeze-dried beads. In one embodiment, one of the immune reaction chambers 115a is pre-loaded with background quality control freeze-dried beads, and the other immune reaction chamber 115a is pre-loaded with reagent degradation quality control freeze-dried beads. In this embodiment, the background quality control freeze-dried beads include freeze-dried beads connected with streptavidin magnetic beads and freeze-dried beads of alkaline phosphatase; the reagent degradation quality control freeze-dried beads include freeze-dried beads of streptavidin magnetic beads and freeze-dried beads of alkaline phosphatase biotin conjugate. It is understood that in other embodiments, the quality control freeze-dried beads can be omitted. Correspondingly, the reaction unit 115 for holding the quality control freeze-dried beads can also be omitted or used to hold allergen freeze-dried beads and labeled antibodies.

[0066] In the illustrated embodiment, the number of reaction units 115 is 27, of which 25 reaction units 115 are used for reacting the serum or plasma of the test sample after separation and treatment of whole blood and dilution with the labeled antibody and different allergen freeze-dried beads, one reaction unit 115 is used for reacting the serum or plasma of the test sample after separation and treatment of whole blood and dilution with the background quality control freeze-dried beads, and one reaction unit 115 is used for reacting the serum or plasma of the test sample after separation and treatment of whole blood and dilution with the reagent degradation quality control freeze-dried beads.

[0067] Specifically, the substrate reagent chamber 116 is used to pre-load the luminescent substrate freeze-dried beads. In the illustrated embodiment, the substrate reagent chamber 116 is located on a side of the blood waste liquid chamber 112c away from the plasma chamber 112a and close to the injection chamber 111. The substrate reagent chamber 116 is connected to the mixing chamber 113 through a microchannel.

[0068] Furthermore, the allergen freeze-dried beads, the labeled antibody freeze-dried beads and the luminescent substrate freeze-dried beads each independently contain a freeze-dried diluent after freeze-drying, and the freeze-dried diluent includes a buffer, an auxiliary agent with a mass percentage of 3% to 20%, a surfactant with a mass percentage of 0.01% to 1% and a preservative with a mass percentage of 0.01% to 0.5% before freeze-drying; the auxiliary agent includes at least one of mannitol, trehalose, sucrose, lactose, dextran, PEG, PVP, BSA and gelatin. The freeze-dried diluent can ensure that the freeze-dried beads have a full and smooth appearance after freeze-drying and have a certain strength and toughness, and make the biologically active components in the freeze-dried beads tolerate storage in a room temperature environment. Optionally, the surfactant includes at least one of Tween 20, Tween 80, Span 80, Triton X-45 and Triton X-100. Optionally, the allergen freeze-dried beads also contain at least one of a protein protectant and an antioxidant; and / or, the labeled antibody freeze-dried beads contain at least one of a protein protectant (e.g., glycerol, calcium chloride, disodium ethylenediaminetetraacetic acid, glycine, etc.) and an antioxidant (e.g., glutathione, ascorbic acid, thioglycerol, cysteine, etc.). Optionally, the buffer is a TBS buffer. It is understood that the buffer and the surfactant are not limited to the above, and the preservatives, protein protectants and antioxidants are not particularly limited. Furthermore, the freeze-dried diluent includes 15% to 20% by weight of an auxiliary agent, 0.5% to 1% by weight of a surfactant, and 0.01% to 0.5% by weight of a preservative before freeze-drying. It should be noted that the allergen freeze-dried beads, the labeled antibody freeze-dried beads and the luminescent substrate freeze-dried beads each independently contain a freeze-dried diluent after freeze-drying, which means that the allergen freeze-dried beads, the labeled antibody freeze-dried beads and the luminescent substrate freeze-dried beads all contain a freeze-dried diluent after freeze-drying, and the respective freeze-dried diluents do not affect each other.

[0069] Specifically, the liquid capsule assembly 120 is located on the cover surface 110a, and is used to provide diluent and cleaning liquid to the chip body 110 to complete the immune response. The liquid capsule assembly 120 includes a plurality of liquid capsules. Optionally, the liquid capsule includes a first liquid capsule 121 storing a plasma diluent, a second liquid capsule 122 storing a cleaning liquid, and a third liquid capsule 123 storing a luminescent substrate diluent. The plasma diluent in the first liquid capsule 121 and the cleaning liquid in the second liquid capsule 122 can flow into the mixing chamber 113, and the luminescent substrate diluent in the third liquid capsule 123 can flow into the mixing chamber 113 through the substrate reagent chamber 116. In some embodiments, the liquid capsule assembly 120 has a first cavity, a second cavity, and a third cavity. The first cavity and the second cavity are respectively connected to the mixing chamber 113, and the third cavity is connected to the mixing chamber 113 through the substrate reagent chamber 116; the first liquid capsule 121 is located in the first cavity, the second liquid capsule 122 is located in the second cavity, and the third liquid capsule 123 is located in the third cavity. It is understandable that in some embodiments, the liquid capsule of the liquid capsule assembly 120 in the microfluidic chip 10 can be omitted, and an additional liquid capsule is required when in use, that is, the corresponding liquid capsule is added to the first cavity, the second cavity, and the third cavity when in use. Of course, in some embodiments, the liquid capsule can also be omitted, and the liquid can be directly added to the corresponding first cavity, the second cavity, and the third cavity, and the corresponding chambers are connected when the corresponding liquid is needed.

[0070] In some embodiments, the liquid capsule assembly 120 also includes a puncturing piece (not shown) corresponding to the liquid capsule. Through the action of external force, the puncturing piece can puncture the liquid capsule corresponding to it so that the liquid stored in the liquid capsule can flow out. In some embodiments, the puncturing piece is located on the chip body 110 and extends from the chip body 110 in the direction close to the liquid capsule. The puncturing piece includes a spike portion, which is used to puncture the liquid capsule. When not in use (when the liquid capsule does not need to be punctured), there is a gap between the puncturing piece and the liquid capsule. It is understandable that in other embodiments, the puncturing piece can be replaced by other switches that can control the opening and closing of the liquid capsule.

[0071] In some embodiments, the microfluidic chip 10 further includes a first puncture member corresponding to the first liquid capsule 121, a second puncture member corresponding to the second liquid capsule 122, and a third puncture member corresponding to the third liquid capsule 123. The first puncture member, the second puncture member, and the third puncture member all have a spike portion, and the shape of the spike portion is a square cone, a cone, and a blade shape. The number of the first puncture member, the second puncture member, and the third puncture member is at least 1 independently. Specifically, the number of second liquid capsules 122 corresponds to the number of cleaning times. For example, when the number of cleaning times is two, the number of second liquid capsules 122 is two. Of course, multiple second liquid capsules 122 are arranged at intervals. It should be noted that the number of cleaning times here refers to the number of times the antigen-antibody complex formed by the capture antigen or capture antibody and the detected substance and the antigen-antibody complex with a label are cleaned. In the illustrated embodiment, the number of the first liquid capsule 121 is one, the number of the second liquid capsule 122 is three, and the number of the third liquid capsule 123 is one. Optionally, the diluent in the first liquid capsule 121 may be the same as the diluent in the third liquid capsule 123 .

[0072] Optionally, the liquid capsule component 120 has a liquid capsule cavity for accommodating the liquid capsule, and the number of the liquid capsule cavity corresponds to the number of the liquid capsules.

[0073] Specifically, the number of the second liquid capsules 122 corresponds to the number of cleaning times. For example, when the number of cleaning times is two, the number of the second liquid capsules 122 is two. Of course, the multiple first liquid capsules 121 are arranged at intervals. It should be noted that the number of cleaning times here refers to the number of times the cleaning allergen or capture antibody forms a complex with the detected substance and the labeled antibody. In the illustrated embodiment, the number of the first liquid capsule 121 is one, the number of the second liquid capsule 122 is three, and the number of the third liquid capsule 123 is one. Optionally, the diluent of the first liquid capsule 121 can be the same as the diluent of the third liquid capsule 123. In some embodiments, the volumes of the first liquid capsule 121, the second liquid capsule 122, and the third liquid capsule 123 are independently 100 μL to 1200 μL.

[0074] Furthermore, the microfluidic chip 10 also includes a cover plate 130, which is covered on the covering surface 110a. The covering of the cover plate 130 and the chip body 110 blocks the openings of each chamber on the chip toward the covering surface 110a, so that the liquid in the chamber will not overflow from the opening and affect the reaction. The first liquid capsule 121, the second liquid capsule 122 and the third liquid capsule 123 are all located on the side of the cover plate 130 away from the chip body 110, and the cover plate 130 is provided with through holes corresponding to the first liquid capsule 121, the second liquid capsule 122 and the third liquid capsule 123 respectively. The through holes provided on the cover plate 130 are used to allow the liquid in the liquid capsule to flow into the mixing chamber 113. In this embodiment, the thickness of the chip body 110 is 2mm to 8mm; the thickness of the cover plate 130 is 0.5mm to 2mm.

[0075] Optionally, the material of the cover plate 130 includes but is not limited to glass, PDMS, PMMA, PET or PC.

[0076] Furthermore, the microfluidic chip 10 further includes an adhesive layer 140 for bonding the cover plate 130 and the chip body 110. In this embodiment, the thickness of the adhesive layer 140 is 0.03 mm to 0.2 mm.

[0077] In addition, an embodiment of the present application also provides a method for preparing the above-mentioned microfluidic chip 10, which includes: obtaining a chip body 110 by injection molding or CNC machining; placing luminescent substrate freeze-dried beads in a substrate reagent chamber 116, labeled antibody freeze-dried beads and different allergen freeze-dried beads in a corresponding immune reaction chamber 115a, background quality control freeze-dried beads in a corresponding immune reaction chamber 115a, and reagent degradation quality control freeze-dried beads in a corresponding immune reaction chamber 115a, and then placing an adhesive layer 140 on the chip body 110; covering the cover plate 130 on the adhesive layer 140 and then pressurizing and bonding them; bonding the first liquid capsule 121, the second liquid capsule 122 and the liquid capsule to corresponding positions of the cover plate 130 to obtain the microfluidic chip 10.

[0078] Optionally, the connection between the cover plate 130 and the chip body 110 is not limited to the connection through the adhesive layer 140 mentioned above, and may also be ultrasonic welding, laser welding, etc. Of course, the adhesive layer 140 and other sealing technologies may also be used simultaneously.

[0079] In one embodiment, the preparation step of the luminescent substrate freeze-dried beads includes: after mixing the luminescent substrate with the freeze-drying diluent, freeze-drying to prepare the luminescent substrate freeze-dried beads. In another embodiment, the preparation step of the luminescent substrate freeze-dried beads includes: adding an auxiliary agent, a surfactant and a preservative to the luminescent substrate solution, then spotting with a liquid nitrogen spotter to prepare frozen microspheres, and then transferring the frozen microspheres to a freeze dryer, and after vacuum freeze drying, a spherical solid luminescent substrate freeze-dried ball is obtained. Of course, the types and amounts of the auxiliary agents, surfactants and preservatives and the freeze-drying diluents are as described above and will not be repeated here.

[0080] In one embodiment, the preparation step of the labeled antibody freeze-dried beads includes: mixing the labeled antibody with a freeze-dried diluent, and then freeze-drying to prepare the labeled antibody freeze-dried beads.

[0081] In one embodiment, the preparation step of the allergen freeze-dried beads comprises: mixing the allergen and the carrier with a freeze-drying diluent respectively, and freeze-drying them respectively to prepare the allergen freeze-dried beads. At this time, the allergen and the carrier exist in the form of freeze-dried beads independent of each other. In another embodiment, the preparation step of the allergen freeze-dried beads comprises: mixing the allergen and the carrier with a freeze-drying diluent after coupling, and then freeze-drying to prepare the allergen freeze-dried beads. At this time, the allergen of the allergen freeze-dried beads has been connected to the carrier.

[0082] In one embodiment, the background quality control freeze-dried beads include magnetic beads freeze-dried beads connected to streptavidin and alkaline phosphatase freeze-dried beads. At this time, the preparation steps of the background quality control freeze-dried beads include: mixing the magnetic beads connected to streptavidin and alkaline phosphatase with freeze-dried diluents, respectively, and then freeze-drying them separately to prepare freeze-dried beads connected to streptavidin magnetic beads and freeze-dried beads of alkaline phosphatase. The reagent degradation quality control freeze-dried beads include streptavidin magnetic beads freeze-dried beads and alkaline phosphatase biotin conjugate freeze-dried beads. At this time, the preparation steps of the reagent degradation quality control freeze-dried beads include: mixing the magnetic beads connected to streptavidin and the alkaline phosphatase biotin conjugate with freeze-dried diluents, respectively, and then freeze-drying them separately.

[0083] In addition, an embodiment of the present application also provides a method for detecting allergens, which uses the above-mentioned microfluidic chip for detecting allergens to perform detection. Specifically, it includes the following steps:

[0084] After the whole blood sample is injected into the injection chamber, the whole blood is separated by the whole blood separation structure to obtain the plasma located in the plasma chamber; the plasma in the plasma chamber is transferred to the mixing chamber; the plasma diluent is introduced into the mixing chamber to dilute the plasma in the mixing chamber; the diluted plasma in the mixing chamber is distributed to each immune reaction chamber through a liquid separation channel; in each immune reaction chamber, the diluted plasma reacts with a pre-installed reagent for immune reaction to form an antigen-antibody complex; after the reaction of each immune reaction chamber is completed, the substances other than the antigen-antibody complex in each immune reaction chamber are transferred to the reaction waste liquid chamber; the cleaning liquid is introduced into each immune reaction chamber through the mixing chamber to clean the antigen-antibody complex formed in each immune reaction chamber after the reaction is completed; after the cleaning is completed, the luminescent substrate diluent is transferred to each immune reaction chamber through the substrate reagent chamber, the mixing chamber, and the liquid separation channel for reaction, so that the marker on the antigen-antibody complex catalyzes the substrate reaction to generate a chemiluminescent signal; and the chemiluminescent signal generated by the labeled antigen-antibody complex is detected.

[0085] Furthermore, in the step of separating whole blood using the whole blood separation structure, the centrifugal speed is 1000rpm to 5000rpm, and the centrifugal time is 90s to 150s; the step of distributing the diluted plasma in the mixing chamber to each immune reaction chamber through the liquid separation channel includes: using a centrifugal speed of 100rpm to 1200rpm to centrifuge for 1s to 60s to distribute the diluted plasma to each cup chamber; and using a centrifugal speed of 1000rpm to 2000rpm to centrifuge for 1s to 60s to distribute the plasma in each cup chamber to each immune reaction chamber; In the step of transferring the substances except the antigen-antibody complex in each immune reaction chamber to the reaction waste liquid chamber, the centrifugal speed is 1000rpm~2000rpm, and the centrifugal time is 1s~60s; in the step of introducing the cleaning solution into each immune reaction chamber through the mixing chamber, the centrifugal speed is 100rpm~1200rpm, and the centrifugal time is 1s~60s; in the step of transferring the luminescent substrate dilution solution to each immune reaction chamber through the substrate reagent chamber, the mixing chamber, and the liquid separation flow channel, the centrifugal speed is 100rpm~1200rpm, and the centrifugal time is 1s~60s.

[0086] Furthermore, the above detection method comprises the following steps:

[0087] S1: injecting a whole blood sample into the injection chamber of the microfluidic chip of any of the above embodiments;

[0088] S2: Transfer the whole blood sample to the sedimentation tank through the microchannel at a centrifugal speed of 1;

[0089] S3: The whole blood sample in the sedimentation tank is transferred to the plasma chamber and the blood cell chamber at a centrifugal speed of 2, and the excess whole blood will enter the collection part and the blood sample sufficient detection chamber through the platform part.

[0090] S4: Centrifuge the whole blood in the plasma chamber and the blood cell chamber at a centrifugal speed of 3 to separate the plasma from the blood cells.

[0091] S5: After the plasma in the plasma chamber enters the mixing chamber through the first siphon channel at a centrifugal speed of 4, the microfluidic chip stops rotating, and an external force ruptures the first liquid capsule, and the diluent A in the first liquid capsule for mixing with the plasma flows into the mixing chamber.

[0092] S6: Mix the plasma and diluent A thoroughly using a mixing centrifuge at a speed of 5.

[0093] S7: Under the condition of centrifugal speed 6, the mixed liquid in the mixing chamber enters the flow channel and the cup separation chamber through the second siphon flow channel.

[0094] S8: Under the condition of centrifugal speed 7, the mixed solution in the cup chamber enters the immune reaction chamber through the first microfluidic channel. After the mixed solution reconstitutes the freeze-dried balls pre-installed in the immune reaction chamber and fully reacts, the magnet rises and absorbs the magnetic particles (magnetic beads) in the mixed solution.

[0095] S9: The liquid in the immune reaction chamber enters the reaction waste liquid chamber through the second microchannel at a centrifugal speed of 8.

[0096] S10: The magnet descends, the microfluidic chip stops rotating, and one of the second liquid capsules is ruptured by external force to release the cleaning liquid in the second liquid capsule. The cleaning liquid enters the mixing chamber through the microchannel, and the liquid in the mixing chamber enters the main channel and the separation cup through the second siphon microchannel at a centrifugal speed of 6.

[0097] S11: After the liquid in the cup chamber enters the immune reaction chamber through the first microchannel at a centrifugal speed of 7, the magnetic beads are fully washed at a centrifugal speed of 9, and then the magnet rises to absorb the magnetic particles in the mixed solution.

[0098] S12: The liquid in the immune reaction chamber enters the reaction waste liquid chamber through the second microchannel at a centrifugal speed of 8.

[0099] S13: The magnet descends, the microfluidic chip stops rotating, and an external force ruptures another second liquid capsule to release the cleaning liquid into the mixing chamber. Then, the cleaning liquid in the mixing chamber enters the main channel and the cup chamber through the second siphon flow channel at a centrifugal speed of 6.

[0100] S14: Repeat the above steps S11 to S13.

[0101] S15: After the other second liquid capsule is ruptured by external force to release the cleaning liquid into the mixing chamber, the cleaning liquid in the mixing chamber is passed through the second siphon flow channel into the main flow channel and the cup separation chamber at a centrifugal speed of 6.

[0102] S16: Repeat the above steps S11 to S13.

[0103] S17: External force ruptures the third liquid capsule to release the diluent into the substrate reagent chamber, re-dissolves the luminescent substrate pre-installed in the substrate reagent chamber and enters the mixing chamber, and at a centrifugal speed of 6, the liquid in the mixing chamber enters the main channel and the cup chamber through the second siphon flow channel.

[0104] S18: Repeat the above steps S11 to S13.

[0105] S19: Collect information from each immune reaction chamber, analyze data, and output results.

[0106] In step S2, the centrifugal speed 1 is 100 to 1200 rpm, and the centrifugal time is 1 to 60 s.

[0107] In step S3, the centrifugal speed 2 is 100 to 1400 rpm, and the centrifugal time is 1 to 60 s.

[0108] In step S4, the centrifugal speed 3 is 1000-5000 rpm, and the centrifugal time is 90s-150s.

[0109] In step S5, the centrifugal speed 4 is 100 to 1200 rpm, and the centrifugal time is 1 to 60 s.

[0110] In step S6, the centrifugal speed 5 is 100 to 4000 rpm, and the centrifugal time is 1 to 60 seconds.

[0111] In step S7, step S10, step S13, step S15 and step S17, the centrifugal speed 6 is 100 rpm to 1200 rpm, and the centrifugal time is 1 s to 60 s.

[0112] In step S8, the centrifugal speed 7 is 1000-2000 rpm, and the centrifugal time is 1 s-60 s.

[0113] In step S9 and step S12, the centrifugal speed 8 is 1000-2500 rpm, and the centrifugal time is 1 s-60 s.

[0114] In step S11, the centrifugal speed 9 is 100 to 1200 rpm, and the centrifugal time is 1 second to 180 seconds.

[0115] It should be noted that in this embodiment, the rotation radius is 60 mm.

[0116] In the above step S19, the micro PMT sequentially locates the reaction chamber corresponding to each item and collects the luminescence intensity RLU and the concentration of the corresponding item and then fits the curve of concentration and luminescence intensity.

[0117] In one embodiment, the curve is a four-parameter fitting curve, which is a nonlinear equation. After establishing a standard curve to obtain the parameters of the curve, the data of each item is analyzed and converted to achieve quantitative or semi-quantitative detection. Further, the specific steps of data analysis include:

[0118] 1. Plot the expected concentration X of the reference product as the abscissa and the corresponding luminescence intensity RLU as the ordinate to obtain the four-parameter fitting curve of each item and its equation parameters, that is, RLU = (ad) / [1+(X / b)^c]+d, where a, b, c, d are equation parameters;

[0119] 2. Subtract the RLU value of the luminescence intensity of the background quality control chamber from the RLU collected by the reaction chamber corresponding to the test item of the target whole blood sample to obtain the net RLU value of the item;

[0120] 3. Subtract the luminescence intensity RLU value of the background quality control chamber from the luminescence intensity RLU value collected by the reagent degradation quality control chamber to obtain the RLU net value of the reagent degradation quality control chamber;

[0121] 4. According to the change of the RLU net value of the reagent degradation quality control chamber, convert the RLU net value of each item, and substitute the converted RLU net value of each item into the equation: X = b × [(ad) / (RLU-d)-1]^(1 / c) to calculate the concentration index corresponding to the test item of the target whole blood sample. Specific embodiments

[0123] The following is a detailed description in conjunction with specific examples. The following examples do not include other components except for inevitable impurities unless otherwise specified. The reagents and instruments used in the examples are conventionally selected in the art unless otherwise specified. The experimental methods for which specific conditions are not specified in the examples are implemented according to conventional conditions, such as the conditions described in the literature, books, or methods recommended by the manufacturer.

[0124] Example 1

[0125] This embodiment prepares a microfluidic chip for detecting inhaled and food allergen-specific IgE antibodies. The structure of the microfluidic chip is as follows: Figure 1 As shown, the specific preparation method of the microfluidic chip includes but is not limited to the following steps:

[0126] 1. Preparation of Biotin Conjugate

[0127] Allergen extracts of house dust mites, house dust, cat hair, dog epithelium, cockroaches, Penicillium, Aspergillus fumigatus, Alternaria alternata, Cladosporium spp., willow, poplar, elm, humulus, mugwort, ragweed, egg white, egg yolk, milk, peanuts, soybeans, beef, mutton, shrimp, crab, cod, lobster, scallop, peach, apple, mango, litchi, strawberry, cashew, pistachio, hazelnut, almond, walnut and CCD, mouse anti-human IgE antibody and alkaline phosphatase were placed in dialysis bags respectively, and dialyzed in PBS buffer overnight; biotin-N-hydroxysuccinimide solution Biotin-NHS was prepared with PBS buffer, 10 μL Biotin-NHS solution was added to every 1 mg antigen (allergen) or antibody, mixed evenly, and reacted at room temperature for 60 minutes; the labeled biotin conjugate was placed in a dialysis bag, and dialyzed in PBS buffer overnight.

[0128] 2. Preparation of Alkaline Phosphatase-labeled Mouse Anti-human IgE Antibody

[0129] A 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid-3-thio-N-succinimidyl ester sodium salt (Sulfo-SMCC) solution was prepared with PB buffer, 0.1 mL of Sulfo-SMCC solution was added to each 1 mg of alkaline phosphatase, mixed evenly, reacted at room temperature for 30 minutes, and dialyzed in PB buffer; a 2-iminothiolane hydrochloride (2-IT) solution was prepared with PB buffer, 0.1 mL of 2-IT was added to each 1 mg of mouse anti-human IgE antibody, mixed evenly, reacted at room temperature for 30 minutes, and dialyzed in PB buffer; the dialyzed alkaline phosphatase and mouse anti-human IgE antibody were mixed evenly, reacted at room temperature for 120 minutes, and alkaline phosphatase-labeled mouse anti-human IgE antibody was obtained.

[0130] 3. Preparation of Lyophilized Dilution

[0131] To TBS buffer, 5% mannitol, 0.5% trehalose, 0.2% sucrose, 2% lactose, 4% PEG, 4% PVP, 0.5% BSA, 0.5% gelatin were added, and then 0.1% Tween 20 was added, and finally 0.01% thimerosal, 0.5% calcium chloride and 0.01% thioglycerol were added to obtain a lyophilized diluent.

[0132] 4. Preparation of Lyophilized Streptavidin Magnetic Beads

[0133] Select streptavidin magnetic beads with a particle size range of 1μm to 5μm to prepare streptavidin magnetic bead freeze-dried balls. Take 10mg of streptavidin magnetic beads and repeat the "magnetic separation-replacement diluent (lyophilized diluent)-mixing" operation steps 3 times, and continue to add 10mL of lyophilized diluent to appropriately dilute the magnetic beads to obtain streptavidin magnetic bead freeze-dried liquid. The streptavidin magnetic bead freeze-dried liquid is sampled by a liquid nitrogen sampler to make frozen microspheres, and then the frozen microspheres are transferred to a freeze dryer. After vacuum freeze drying, spherical solid streptavidin magnetic bead freeze-dried balls are obtained.

[0134] 5. Preparation of Biotin Conjugate Lyophilized Spheres

[0135] The biotin conjugate prepared in step 1 is diluted with the lyophilized diluent prepared in step 3 at a concentration of 1 μg / mL to prepare a biotin conjugate lyophilized solution. The biotin conjugate lyophilized solution is sampled by a liquid nitrogen sampler to prepare frozen microspheres, and then the frozen microspheres are transferred to a freeze dryer. After vacuum freeze drying, spherical solid biotin conjugate lyophilized beads are obtained.

[0136] 6. Preparation of Lyophilized Alkaline Phosphatase Pellets

[0137] The alkaline phosphatase lyophilization diluent was diluted to a concentration of 0.5 μg / mL to prepare an alkaline phosphatase lyophilized solution. The alkaline phosphatase lyophilized solution was sampled by a liquid nitrogen sampler to prepare frozen microspheres, and then the frozen microspheres were transferred to a freeze dryer, and after vacuum freeze drying, spherical solid alkaline phosphatase lyophilized beads were obtained.

[0138] 7. Preparation of freeze-dried pellets of mouse anti-human IgE antibody labeled with alkaline phosphatase

[0139] The alkaline phosphatase-labeled mouse anti-human IgE antibody was diluted with the lyophilized diluent prepared in step 3 at a concentration of 0.5 μg / mL to prepare an alkaline phosphatase-labeled mouse anti-human IgE antibody lyophilized liquid. The alkaline phosphatase-labeled mouse anti-human IgE antibody lyophilized liquid was spotted with a liquid nitrogen spotter to prepare frozen microspheres, and then the frozen microspheres were transferred to a freeze dryer, and after vacuum freeze drying, spherical solid alkaline phosphatase-labeled mouse anti-human IgE antibody lyophilized beads were obtained.

[0140] 8. Preparation of luminescent substrate freeze-dried beads

[0141] 12% mannitol, 5% lactose, 3% PVP and 0.01% thimerosal were added to the luminescent substrate solution, and finally the frozen microspheres were prepared by spotting with a liquid nitrogen spotter. The frozen microspheres were then transferred to a freeze dryer and vacuum freeze-dried to obtain spherical solid luminescent substrate freeze-dried beads.

[0142] 9. Chip assembly:

[0143] The chip body 110 is obtained by injection molding or machining. 25 of the 27 immune reaction chambers 114a are filled with allergen extracts of various items (1 house dust mite (number "1" represents the number, the same below), 2 dust mites, 3 house dust, 4 cat hair, 5 dog epithelium, 6 cockroaches, 7 Penicillium / Aspergillus fumigatus / Alternariae / Cladosporium, 8 willow / poplar / elm, 9 Humulus, 10 Artemisia, 11 ragweed, 12 egg white, 13 egg yolk, 14 milk, 15 peanuts, 16 soybeans, 17 beef, 18 mutton, 19 shrimp, 20 crab, 21 cod / lobster / scallop, 22 peach / apple / mango / lychee / strawberry, 23 cashew / pistachio / hazelnut / almond / walnut, 24 CCD) and 25 mouse anti-human IgE antibody biotin conjugate freeze-dried ball, streptavidin magnetic bead freeze-dried ball and alkaline phosphatase labeled mouse anti-human IgE antibody freeze-dried ball; one of the remaining two immune reaction chambers 114a is a background quality control chamber, loaded with streptavidin magnetic bead freeze-dried ball and alkaline phosphatase freeze-dried ball, and the other is a reagent degradation quality control chamber, loaded with streptavidin magnetic bead freeze-dried ball and alkaline phosphatase biotin conjugate freeze-dried ball. The substrate reagent chamber 116 is loaded with luminescent substrate freeze-dried ball. Then, the adhesive layer 140 is covered on the chip body 110 by a sealing device, and the cover plate 130 is covered on the adhesive layer 140 to form a combined chip. A certain pressure is applied to the combined chip so that the three are tightly bonded together. Finally, the first liquid capsule 121, the second liquid capsule 122, and the third liquid capsule 123 with glue are pasted to the corresponding positions and pressed tightly. At this point, the microfluidic chip 10 for detecting inhaled and food allergen-specific IgE antibodies is completed.

[0144] Example 2

[0145] The inhalation and food allergen-specific IgE antibody chip prepared in Example 1 was used to test 11 inhalation group allergen-specific IgE antibodies and 12 food group allergen-specific IgE antibodies and total IgE antibodies in 20 whole blood samples. The specific steps of testing each sample include:

[0146] 1. Inject 500 μL of blood sample into the injection chamber 111 of the microfluidic chip 10, and then place the microfluidic chip 10 into a detection machine that matches the chip.

[0147] 2. The machine first performs centrifugation at speed 1 (100 rpm) to transfer the blood sample to the sedimentation tank 117 through the microchannel.

[0148] 3. Then, centrifuge at speed 2 (150 rpm) to transfer the blood sample to the plasma chamber 112a. Excess blood will pass through the platform portion 112d and enter the collection portion 112e and the blood sample sufficient amount detection chamber 112f.

[0149] 4. The machine executes high-speed centrifugation speed 3 (1000 rpm) to centrifuge the whole blood in the plasma chamber 112a for a set time, and the plasma is separated from the blood cells.

[0150] 5. The machine performs low-speed centrifugation at speed 4 (100 rpm). At speed 4, the plasma in the plasma chamber 112a enters the mixing chamber 113 through the first siphon channel 118; at the same time, the machine's rupture mechanism ruptures the first liquid capsule 121 containing the diluent A located above the liquid capsule inlet. The diluent A in the first liquid capsule 121 flows into the mixing chamber 113 through the microchannel.

[0151] 6. After a certain period of time, the machine performs a mixing centrifugation speed of 5 (150rpm). At this speed, the plasma and diluent A are fully mixed.

[0152] 7. At the centrifugal speed of 6 (100 rpm), the mixed liquid in the mixing chamber 113 enters the main channel 114a through the second siphon channel 119 and enters the cup-distributing chamber 114b at the same time.

[0153] 8. After the equal division is completed, the machine performs centrifugal speed 7 (1050rpm), and the mixed solution in the cup chamber 114b enters the immune reaction chamber 115a through the first microchannel 115c. At this time, the mixed solution re-dissolves the freeze-dried beads pre-installed in the immune reaction chamber 115a. The two are fully reacted for 10 minutes. At this time, the magnet in the machine rises and absorbs the magnetic beads in the mixed solution.

[0154] 9. The machine performs centrifugal speed 8 (1050 rpm), and the liquid in the immune reaction chamber 115a enters the reaction waste liquid chamber 115b through the second microchannel 115d. At this time, the system completes one round of reaction.

[0155] 10. The rupture mechanism ruptures the second liquid capsule 122 near the first liquid capsule 121, and the diluent B enters the mixing chamber 113 through the microchannel. The machine performs centrifugal speed 6. The reagent in the mixing chamber 113 enters the main channel 114a through the second siphon channel 119, and enters the cup chamber 114b at the same time.

[0156] 11. After the equal division is completed, the machine performs centrifugal speed 7, and the mixed solution in the cup chamber 10013 enters the immune reaction chamber 115a through the first microchannel 115c. The machine performs a cleaning centrifugal speed 9 (100rpm), and the cleaning solution fully cleans the magnetic beads. Then the magnet in the machine rises and absorbs the magnetic particles in the mixed solution.

[0157] 12. The machine performs centrifugal rotation speed 8, and the liquid in the immune reaction chamber 115a enters the reaction waste liquid chamber 115b through the second microchannel 115d. At this time, the system completes another round of reaction.

[0158] 13. The rupture mechanism ruptures the other second liquid capsule 122, and the diluent B enters the mixing chamber 113 through the microchannel. The machine performs centrifugal speed 6. The reagent in the mixing chamber 113 enters the main channel 114a through the second siphon channel 119, and enters the cup chamber 114b at the same time.

[0159] 14. Repeat the above steps 11 to 13 to complete another round of reaction.

[0160] 15. The rupture mechanism ruptures the remaining second liquid capsule 122, and the diluent B enters the mixing chamber 113 through the microchannel. The machine performs centrifugal speed 6. The reagent in the mixing chamber 113 enters the main channel 114a through the second siphon channel 119, and enters the cup chamber 114b at the same time.

[0161] 16. Repeat the above steps 11 to 13 to complete another round of reaction.

[0162] 17. The rupture mechanism ruptures the third liquid capsule 123, and the diluent A enters the substrate reagent chamber 116. The machine performs a mixing centrifugation speed of 5 to re-dissolve the luminescent substrate freeze-dried beads pre-installed there, and the liquid then enters the mixing chamber 113. The machine performs a centrifugation speed of 6. The reagent in the mixing chamber 113 enters the main channel 114a through the second siphon flow channel 119, and enters the cup chamber 114b at the same time.

[0163] 18. After the equal division is completed, the machine performs centrifugation at a speed of 7, and the mixed solution in the cup chamber 114b enters the immune reaction chamber 115a through the first microchannel 115c. The machine performs a washing centrifugation at a speed of 9, and maintains the incubation reaction for 1 min.

[0164] 19. After the reaction, the machine rotates the disk and positions the micro PMT in each reaction chamber in turn and collects luminescence intensity data. The instrument background analyzes the data and outputs the concentration results of each item. The results are shown in Table 1.

[0165] Table 1

[0166]

[0167]

[0168] As can be seen from Table 1, the inhalation and food allergen specific IgE antibody chip prepared in Example 1 can detect specific IgE antibodies and total IgE antibodies of 11 inhalation allergens and 12 food allergens in whole blood samples. Through the above test results, clinicians can combine the patient's medical history, physical signs, in vivo tests and other clinical information as an auxiliary means to diagnose IgE-mediated allergic diseases.

[0169] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0170] The above-described embodiments only express several implementation methods of the present invention, which are convenient for understanding the technical solutions of the present invention in detail, but they cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the protection scope of the claims attached to the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the contents of the attached claims, and the description and drawings can be used to interpret the contents of the claims.

Claims

1. A microfluidic chip for detecting allergens, characterized in that: include: A chip body, the chip body having a rotation center, and the chip body is provided with: Injection cavity; A whole blood separation structure, comprising a plasma chamber, a blood cell chamber and a blood waste liquid chamber, wherein the plasma chamber is connected to the sample injection chamber, the blood cell chamber and the blood waste liquid chamber are respectively connected to the plasma chamber, and the blood cell chamber is further away from the rotation center than the plasma chamber; A mixing chamber, connected to the plasma chamber; An arc-shaped liquid separation channel, the liquid separation channel comprises a main channel and a plurality of cup-separating cavities, the main channel has an inlet end and an outlet end, the inlet end is connected to the mixing cavity; the main channel is arc-shaped, the plurality of cup-separating cavities are arranged at intervals in the circumferential direction of the main channel and are connected to the main channel, and the distance from the main channel to the rotation center gradually increases from the inlet end to the outlet end; A plurality of reaction units, wherein the plurality of reaction units are spaced apart along the circumferential direction of rotation, wherein the reaction units include an immune reaction chamber and a reaction waste liquid chamber connected to the immune reaction chamber, wherein the immune reaction chamber is correspondingly connected to the cup chamber, and the cup chamber, the immune reaction chamber and the reaction waste liquid chamber are sequentially arranged along the radial direction of the main channel away from the rotation center; allergen freeze-dried beads and labeled antibody freeze-dried beads are pre-loaded in some of the immune reaction chambers, wherein the allergen freeze-dried beads include allergens and carriers, and the allergens in each immune reaction chamber are different; A substrate reagent chamber pre-loaded with luminescent substrate freeze-dried beads, the substrate reagent chamber being connected to the mixing chamber; A liquid capsule component, the liquid capsule component includes a first liquid capsule storing a plasma diluent, a second liquid capsule storing a cleaning liquid, and a third liquid capsule storing a luminescent substrate diluent, the plasma diluent in the first liquid capsule can enter the mixing chamber, the cleaning liquid in the second liquid capsule can enter the mixing chamber, and the luminescent substrate diluent in the third liquid capsule can enter the mixing chamber through the substrate reagent chamber.

2. The microfluidic chip according to claim 1, characterized in that: There are at least 12 reaction units; each of the allergens is independently derived from the following group: house dust mites; dust mites; house dust; cat hair; dog hair; cockroaches; mold; pollen; humulum; mugwort; ragweed; egg white; egg yolk; milk; peanuts; soybeans; beef; mutton; seafood; allergenic fruits; nuts.

3. The microfluidic chip according to claim 2, characterized in that: The allergenic fruits include at least one of peach, apple, mango, lychee and strawberry; and / or, the pollen includes at least one of willow pollen, poplar pollen and elm pollen; and / or, the seafood includes at least one of shrimp, crab, cod and scallop; and / or, the nuts include at least one of cashew, pistachio, hazelnut, almond and walnut; and / or, the mold includes at least one of Penicillium, Aspergillus fumigatus, Alternaria alternata and Cladosporium.

4. The microfluidic chip according to claim 1, characterized in that: Some of the immune reaction chambers are pre-loaded with background quality control freeze-dried beads, and some of the immune reaction chambers are pre-loaded with reagent degradation quality control freeze-dried beads; The background quality control freeze-dried beads include magnetic freeze-dried beads connected with streptavidin and alkaline phosphatase freeze-dried beads.

5. The microfluidic chip according to claim 1, characterized in that: The carrier and the allergen can be coupled via avidin and biotin.

6. The microfluidic chip according to claim 1, characterized in that: The carrier is a magnetic bead connected with streptavidin, and the allergen is coupled with biotin.

7. The microfluidic chip according to any one of claims 1 to 6, characterized in that: The allergen freeze-dried beads, the labeled antibody freeze-dried beads and the luminescent substrate freeze-dried beads each independently contain a freeze-dried diluent after freeze-drying, and the freeze-dried diluent before freeze-drying includes a buffer, an auxiliary agent with a weight percentage of 3% to 20%, a surfactant with a weight percentage of 0.01% to 1% and a preservative with a weight percentage of 0.01% to 0.5%; the auxiliary agent includes at least one of mannitol, trehalose, sucrose, lactose, dextran, PEG, PVP, BSA and gelatin.

8. The microfluidic chip according to claim 7, characterized in that: The surfactant includes at least one of Tween 20, Tween 80, Span 80, Triton X-45 and Triton X-100; And / or, the buffer is TBS buffer; Furthermore, the allergen freeze-dried beads also contain at least one of a protein protectant and an antioxidant; and / or the labeled antibody freeze-dried beads contain at least one of a protein protectant and an antioxidant.

9. The microfluidic chip according to any one of claims 1 to 6 and 8, characterized in that: The liquid capsule component has a first cavity, a second cavity and a third cavity. The first cavity and the second cavity are respectively connected to the mixing cavity. The third cavity is connected to the mixing cavity via the substrate reagent cavity. The first liquid capsule is located in the first cavity, the second liquid capsule is located in the second cavity, and the third liquid capsule is located in the third cavity.

10. A method for detecting an allergen, characterized in that: The detection is performed using the microfluidic chip described in any one of claims 1 to 9.

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