Centrifugal microfluidic chip for allergen detection and methods of use thereof

Abstract

The application discloses a centrifugal microfluidic chip for allergen detection, comprising a chip main body and a plurality of microstructures, wherein the chip main body has a rotation center, and the microstructures comprise: a waste chamber located at the farthest end of the rotation center; a reaction detection chamber provided with a carrier, the carrier being coated with anti-human IgE antibodies; a circular truncated cone-shaped channel, the narrow end of which is connected with the other end of the reaction detection chamber, and the wide end of which is connected with a main channel; a serum separation structure comprising a blood cell chamber and a serum chamber, the serum chamber being connected with the main channel through a siphon valve; a first buffer solution chamber; an allergen chamber; a second buffer solution chamber; an enzyme storage chamber; a third buffer solution chamber; a bottom liquid chamber; and a termination liquid chamber. The application avoids contamination and evaporation of reagents, does not need additional driving equipment, is conducive to integration and miniaturization of the equipment, saves consumption of sample whole blood and reagents, reduces cost and time, and is simple to operate; the application needs small amounts of sample and reagents, has low cost, short detection time, and high detection sensitivity.

Description

Technical Field

[0001] This invention relates to the field of microfluidic chip technology, and in particular to a centrifugal microfluidic chip for allergen detection and its method of use. Background Technology

[0002] Currently, with the increasing severity of global environmental pollution and changes in human lifestyles and medication practices, the number of allergic individuals worldwide is growing, and the incidence of allergic diseases is increasing year by year. The incidence is increasingly seen in young children, and cases of multiple allergens and multi-organ allergies are becoming more common. However, in many cases, the source of the allergen is unknown. Therefore, early detection of allergens can help patients avoid repeated exposure and receive timely treatment, greatly improving their quality of life. Existing in vitro allergen detection methods include: Radioactive Allergen Adsorption Assay (RAST), Western blotting, fluorescence enzyme-linked immunosorbent assay (ELISA), ELISA capture assay, ELISA (microplate assay), chemiluminescence immunoassay, colloidal gold assay, lateral flow immunochromatography (LFIA), allergen microarray assay, and flow cytometry. However, these methods suffer from drawbacks such as high cost, long testing time, large required serum sample volume, excessive reagent consumption, and environmental pollution. In contrast, microfluidic technology combined with immunoassay offers advantages such as requiring less sample and reagent volume and eliminating the need for whole blood separation in vitro. Therefore, it is necessary to design an allergen detection chip based on microfluidic technology. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the present invention aims to provide a centrifugal microfluidic chip for allergen detection and its usage method.

[0004] To achieve the above objectives, an embodiment of the present invention provides the following technical solution:

[0005] A centrifugal microfluidic chip for allergen detection includes a chip body and a plurality of microstructures disposed circumferentially on the chip body. The chip body has a center of rotation, and the microstructures include:

[0006] The waste chamber is located at the farthest end of the rotation center;

[0007] A reaction detection chamber is provided in which a carrier is coated with anti-human IgE antibodies. One end of the reaction detection chamber is connected to the waste chamber through a first burst valve.

[0008] A frustum-shaped channel, the narrow end of which is connected to the other end of the reaction detection chamber, and the wide end of which is connected to the main channel;

[0009] A serum separation structure, comprising a blood cell chamber and a serum chamber connected to the blood cell chamber, wherein the serum chamber is connected to the main channel via a siphon valve;

[0010] The first buffer chamber is connected to the main channel via at least one second burst valve;

[0011] An allergen chamber, which is connected to the main channel via a third burst valve, is used to pre-load allergen-biotin;

[0012] The second buffer chamber is connected to the main channel via at least one fourth burst valve;

[0013] An enzyme storage chamber, which is connected to the main channel via a fifth burst valve, is used to pre-fill horseradish peroxidase-streptavidin conjugate solution;

[0014] The third buffer chamber is connected to the main channel via at least one sixth burst valve;

[0015] The bottom liquid chamber is connected to the main channel via a seventh rupture valve and is used to pre-fill tetramethylbenzidine solution.

[0016] The termination liquid chamber is connected to the main channel via an eighth rupture valve and is used to pre-fill sulfuric acid solution.

[0017] As a further improvement of the present invention, the carrier is a polystyrene ball with a diameter of 115-150 μm.

[0018] As a further improvement of the present invention, a first microchannel is connected between one end of the reaction detection chamber and the waste chamber, the first rupture valve is connected to the first microchannel, and a second microchannel is connected between the narrow end of the frustum-shaped channel and the other end of the reaction detection chamber.

[0019] As a further improvement of the present invention, the width of the first microchannel and the width of the second microchannel are both 90-110μm.

[0020] As a further improvement of the present invention, the angle between the generatrix of the frustum-shaped channel and its center line is 20°-50°.

[0021] As a further improvement of the present invention, the width of the main channel is 500-800μm.

[0022] As a further improvement of the present invention, the blood cell chamber is circular, and / or the serum chamber is fan-shaped.

[0023] As a further improvement of the present invention, the diameters of the first rupture valve, the third rupture valve, the fifth rupture valve, the seventh rupture valve, and the eighth rupture valve are all 1.6-2.6 mm.

[0024] As a further improvement of the present invention, the diameters of the second rupture valve, the fourth rupture valve, and the sixth rupture valve are all 0.8-1.3 mm.

[0025] A method of using a centrifugal microfluidic chip for allergen detection, comprising the following steps:

[0026] (1) A whole blood sample is added to the serum separation structure, and the separated serum enters the reaction detection chamber;

[0027] (2) Release the washing buffer in the first buffer chamber into the reaction detection chamber for cleaning;

[0028] (3) Release the allergen-biotin in the allergen chamber into the reaction detection chamber;

[0029] (4) Release the washing buffer in the second buffer chamber into the reaction detection chamber for cleaning;

[0030] (5) Release the horseradish peroxidase-streptavidin conjugate solution in the enzyme storage chamber into the reaction detection chamber;

[0031] (6) Release the washing buffer in the third buffer chamber into the reaction detection chamber for cleaning;

[0032] (7) Release the tetramethylbenzidine solution in the bottom liquid chamber into the reaction detection chamber;

[0033] (8) Release the sulfuric acid solution in the termination chamber into the reaction detection chamber;

[0034] (9) After the reaction is completed, the solution in the reaction detection chamber is detected and analyzed.

[0035] The beneficial effects of this invention are:

[0036] (1) The present invention prevents the reagent from coming into contact with the external environment, thus avoiding contamination and evaporation of the reagent.

[0037] (2) The present invention utilizes burst valves and siphon valves, which do not require additional driving equipment. The valves can be opened simply by changing the rotation speed of the disc chip, making the microfluidic chip more conducive to the integration and miniaturization of the equipment.

[0038] (3) The present invention integrates a serum separation structure, eliminating the need for separate pretreatment of whole blood samples, saving the consumption of whole blood and reagents, reducing costs and shortening time. All reagents are pre-loaded onto the microfluidic chip, making its operation simple.

[0039] (4) It can remove the interference of non-specific antibodies in serum, greatly improve the detection sensitivity, and does not require surface modification of the chip.

[0040] (5) Based on microfluidic technology combined with immunoassay, it has the advantages of requiring less sample and reagent volume, lower cost, shorter detection time, and higher detection sensitivity. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0042] Figure 1 This is a schematic diagram of the overall structure of a preferred embodiment of the present invention;

[0043] Figure 2 This is a schematic diagram of the microstructure disposed on the chip body according to a preferred embodiment of the present invention;

[0044] Figure 3 This is an enlarged schematic diagram of the structure connecting the frustum-shaped channel to the first burst valve;

[0045] Figure 4 This is a force diagram of the liquid on the capillary burst valve according to a preferred embodiment of the present invention;

[0046] Figure 5 This is a flowchart illustrating the detection process of a centrifugal microfluidic chip according to a preferred embodiment of the present invention.

[0047] Figure 6 This is a standard curve of OD versus total IgE concentration according to a preferred embodiment of the present invention;

[0048] Figure 7 This is a grading standard diagram for serum sIgE concentration detection according to a preferred embodiment of the present invention;

[0049] In the diagram: 1. Chip body; 100. Microstructure; 101. Rotation center; 2. Waste chamber; 3. Reaction detection chamber; 4. Frustum-shaped channel; 5. Main channel; 6. Serum separation structure; 7. First buffer chamber; 8. Allergen chamber; 9. Second buffer chamber; 10. Enzyme storage chamber; 11. Third buffer chamber; 12. Base fluid chamber; 13. Termination fluid chamber; 21. First burst valve; 22. Second burst valve; 23. Third burst valve; 24. Fourth burst valve; 25. Fifth burst valve; 26. Sixth burst valve; 27. Seventh burst valve; 28. Eighth burst valve; 31. Siphon valve; 41. First microchannel; 42. Second microchannel; 601. Blood cell chamber; 602. Serum chamber; 603. Third microchannel. Detailed Implementation

[0050] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0051] Please see Figures 1-3 This application discloses a centrifugal microfluidic chip for allergen detection, including a chip body 1 and a plurality of microstructures 100 disposed circumferentially on the chip body 1. The chip body has a rotation center 101, and the microstructures 100 include:

[0052] Waste chamber 2 is located at the farthest end of the rotation center 101;

[0053] The reaction detection chamber 3 contains a carrier coated with anti-human IgE antibodies. One end of the reaction detection chamber 3 is connected to the waste chamber 2 via a first burst valve 21.

[0054] The narrow end of the frustum-shaped channel 4 is connected to the other end of the reaction detection chamber 3, and the wide end of the frustum-shaped channel 4 is connected to the main channel 5.

[0055] The serum separation structure 6 includes a blood cell chamber 601 and a serum chamber 602 connected to the blood cell chamber 601. The serum chamber 602 is connected to the main channel 5 through a siphon valve 31.

[0056] The first buffer chamber 7 is connected to the main channel 5 via at least one second burst valve 22;

[0057] Allergen chamber 8 is connected to the main channel via a third burst valve 23. Allergen chamber 8 is used to pre-load allergen-biotin.

[0058] The second buffer chamber 9 is connected to the main channel 5 via at least one fourth burst valve 24.

[0059] Enzyme storage chamber 10 is connected to the main channel 5 via the fifth burst valve 25. Enzyme storage chamber 10 is used to pre-load horseradish peroxidase-streptavidin conjugate solution.

[0060] The third buffer chamber 11 is connected to the main channel 5 via at least one sixth burst valve 26;

[0061] The bottom liquid chamber 12 is connected to the main channel 5 via the seventh rupture valve 27. The bottom liquid chamber 12 is used to pre-fill tetramethylbenzidine solution.

[0062] Termination chamber 13 is connected to main channel 5 via eighth rupture valve 28. Termination chamber 13 is used to pre-fill sulfuric acid solution.

[0063] The rotation center 101 of the chip body 1 is used to mount the rotating part of the rotating device, serving as the rotation center during centrifugal operation. Preferably, the chip body 1 is disc-shaped. Preferably, the chip body 1 is made of PMMA, is a transparent material, and has a diameter of 145mm-170mm. More preferably, the diameter of the chip body 1 is 150mm. The chip body 1 is manufactured by injection molding. The chip body 1 includes a base plate and a top plate. The base plate has the following components: waste chamber 2, reaction detection chamber 3, frustum-shaped channel 4, main channel 5, serum separation structure 6, first burst valve 21, second burst valve 22, third burst valve 23, fourth burst valve 24, fifth burst valve 25, sixth burst valve 26, seventh burst valve 27, eighth burst valve 28, siphon valve 31, first buffer chamber 7, second buffer chamber 9, third buffer chamber 11, allergen chamber 8, enzyme storage chamber 10, base liquid chamber 12, and stop liquid chamber 13. The top plate has sample loading holes, ventilation holes, etc.

[0064] The preferred carrier is polystyrene spheres with a diameter of 115-150 μm. More preferably, the diameter of the polystyrene spheres is 130 μm. Because the polystyrene spheres are coated with anti-human IgE antibodies, they can capture specific IgE antibodies in serum, removing interference from non-specific antibodies and significantly improving detection sensitivity. The carrier is not limited to polystyrene spheres; it can also be silica beads, magnetic beads, polymethyl methacrylate microbeads, agarose beads, etc.

[0065] Preferably, one end of the reaction detection chamber 3 is connected to the waste chamber 2 via a first microchannel 41, and a first burst valve 21 is connected to the first microchannel 41. The narrow end of the frustum-shaped channel 4 is connected to the other end of the reaction detection chamber 3 via a second microchannel 42. The wide end of the frustum-shaped channel 4 is connected to the main channel 5. By setting the frustum-shaped channel 4, the transition of liquid from the main channel 5 to the second microchannel 42 is facilitated.

[0066] Preferably, the width of the first microchannel 41 and the width of the second microchannel 42 are both 90-110 μm. More preferably, the width of the first microchannel 41 and the width of the second microchannel 42 are both 100 μm.

[0067] Preferably, the angle between the generatrix of the frustum-shaped channel 4 and its centerline is 20°-50°, achieving a transition from the wider main channel 5 to the narrower second microchannel 42. More preferably, the angle between the generatrix of the frustum-shaped channel 4 and its centerline is 45°, providing an even better transition.

[0068] The width of the main channel 5 is preferably 500-800 μm. More preferably, the width of the main channel 5 is 600 μm.

[0069] Preferably, the blood cell chamber 601 is circular, and the serum chamber 602 is fan-shaped. Since serum separation relies on centrifugal force, which pushes blood cells in whole blood towards the edge of the chip and stores them in the blood cell chamber 601, the circular shape of the blood cell chamber 601 provides a larger surface area for the blood cells and facilitates faster sedimentation. The fan-shaped structure, with a uniform radius at the bottom, ensures that the applied centrifugal force is constant along the entire length of the chamber, further promoting the movement of blood cells into the blood cell chamber 601, improving serum purity, and reducing separation time. A third microchannel 603 connects the blood cell chamber 601 and the serum chamber 602 to prevent remixing of the separated blood cells and serum. Preferably, the third microchannel 603 has a width of 400-1200 micrometers, a length of 1.5-2 millimeters, and a depth of 300-600 micrometers. Preferably, the third microchannel 603 has a width of 500 micrometers, a length of 1.8 millimeters, and a depth of 400 micrometers. Preferably, the third microchannel 603 is higher than the serum chamber 602 and the blood cell chamber 601, so that the third microchannel 603 acts as a microvalve, preventing the backflow of separated blood cells, that is, preventing the separated blood cells and serum from remixing. Preferably, the third microchannel 603 is 600 micrometers higher than the serum chamber 602 and the blood cell chamber 601.

[0070] The working principle of the siphon valve 31 is that when the rotation speed is reduced to a certain range, the capillary force overcomes the centrifugal force and drives the liquid to flow inward through the siphon wave peak and fill the entire channel; then, as the rotation speed increases again, the hydrostatic pressure generated by centrifugation discharges the fluid through the siphon channel to the outlet chamber, thereby realizing the transfer of the serum obtained by high-speed centrifugation to the reaction detection chamber 3 through the siphon valve 31.

[0071] Preferably, the diameters of the first rupture valve 21, the third rupture valve 23, the fifth rupture valve 25, the seventh rupture valve 27, and the eighth rupture valve 28 are all 1.6-2.6 mm. More preferably, the diameters of the first rupture valve 21, the third rupture valve 23, the fifth rupture valve 25, the seventh rupture valve 27, and the eighth rupture valve 28 are all 1.9 mm.

[0072] Preferably, the diameters of the second rupture valve 22, the fourth rupture valve 24, and the sixth rupture valve 26 are all 0.8-1.3 mm. More preferably, the diameters of the second rupture valve 22, the fourth rupture valve 24, and the sixth rupture valve 26 are all 1 mm.

[0073] The first buffer chamber 7, the second buffer chamber 9, and the third buffer chamber 11 are all pre-filled with Tween phosphate buffer. Preferably, the first buffer chamber 7 is connected to the main channel 5 via three second burst valves 22. The positions of the three second burst valves 22 divide the volume of the first buffer chamber 7 into three equal parts, ensuring that each burst valve 22 releases the same volume of washing buffer upon opening. Three washes thoroughly flush the residual liquid from reagent hybridization in the reaction detection chamber 3, reducing false positives and improving detection accuracy. Preferably, the second buffer chamber 9 is connected to the main channel 5 via three fourth burst valves 24. Preferably, the third buffer chamber 11 is connected to the main channel 5 via three sixth burst valves 26.

[0074] The following analysis examines the forces acting on the liquid in the capillary burst valve.

[0075] In a microfluidic centrifugal disk platform, centrifugal force provides the pumping pressure, while capillary force at the connection inhibits flow. This constitutes a capillary rupture valve dependent on the disk's rotational speed to control fluid release and flow. Therefore, the fluid is subjected to both centrifugal and capillary forces... Figure 4 As shown, where, Figure 4 Figure 4(a) shows the distance from the center of the disk to the liquid element at different locations. Figure 4(b) shows a two-dimensional force analysis diagram of the liquid at the connection between the microchannel and the capillary burst valve. Figure 4(c) shows a three-dimensional force analysis diagram of the liquid at the connection between the microchannel and the capillary burst valve. The pumping force P per unit area generated by centrifugal force... c The formula is Where ρ is the density of the liquid, ω is the angular velocity of the disk, and r is the distance from a liquid element to the center of the disk. The integral from R1 to R2 is then... Where ΔR equals R2-R1. equal R1 and R2 are two distances from the liquid to the center of the disk. For microfluidic centrifugal disk platforms, it is crucial to deliver the solution from each reservoir in a pre-defined manner. For this purpose, a capillary rupture valve is incorporated into the design of the microfluidic centrifugal disk platform. As fluid reaches the interface through the microchannels, the capillary force at the microchannel ends tends to retain the fluid due to the change in geometry. The capillary force P per unit area caused by surface tension is... s For ΔP s = (Cγsinθ) / A, where γ is the surface tension of the fluid, θ is the contact angle, A is the cross-sectional area of ​​the microchannel at the valve connection, and C is the perimeter of the relevant contact line of the microchannel at the valve connection. The burst frequency is defined as ΔP c Greater than or equal to ΔP s The angular frequency at that rotational speed. At this rotational speed, the liquid overcomes the capillary force ΔP. s The resulting pressure flows through the capillary rupture valve, releasing liquid from the reservoir. This is due to the pressure generated by ΔP. c and ΔP s The two formulas can be used to calculate the burst frequency f. b for in, d H W and H represent the hydrodynamic diameter of the connection channel, and the width and height of the valve connection, respectively. As can be seen from the above, the capillary rupture valve is a passive valve, requiring no additional moving parts. It is controlled by the angular velocity of rotation, fluid density, surface tension, and the geometry and position of the channel and reservoir.

[0076] In this embodiment, with the rotation center 101 of the chip body 1 as the center, preferably the waste chamber 2, the first burst valve 21, the reaction detection chamber 3, the frustum-shaped channel 4, the main channel 5, the eighth burst valve 28, and the termination liquid chamber 13 are all on the same radial direction, with the termination liquid chamber being closest to the rotation center 101. The serum separation structure 6, the allergen chamber 8, the second buffer chamber 9, and the bottom liquid chamber 12 are located on one side of this radial direction, and the distance between the serum separation structure 6, the allergen chamber 8, the second buffer chamber 9, and the bottom liquid chamber 12 and the rotation center 101 gradually decreases. The first buffer chamber 7, the enzyme storage chamber 10, and the third buffer chamber 11 are located on the other side of this radial direction, and the distance between the first buffer chamber 7, the enzyme storage chamber 10, and the third buffer chamber 11 and the rotation center 101 gradually decreases.

[0077] This centrifugal microfluidic chip for allergen detection uses polystyrene spheres coated with anti-human IgE antibodies as a carrier and places them in the reaction chamber 3 to capture sIgE in serum. This can remove the interference of non-specific antibodies in serum, significantly improve detection sensitivity, and eliminate the need for surface modification of the chip body 1. At the same time, this microfluidic chip has the characteristics of integration and miniaturization, which can solve the shortcomings of large-scale equipment such as long detection time, high price, large required serum sample volume, and excessive reagent consumption. In addition, using the centrifugal microfluidic chip for allergen detection can save the consumption of whole blood samples and reagents, reduce costs, and shorten the time.

[0078] This application also discloses a method for using a centrifugal microfluidic chip for allergen detection. Using the centrifugal microfluidic chip described above includes the following steps:

[0079] S1: Add whole blood sample to serum separation structure 6;

[0080] S2: Release the washing buffer from the first buffer chamber 7 for cleaning;

[0081] S3: Releases the allergen-biotin from allergen chamber 8;

[0082] S4: Release the washing buffer from the second buffer chamber 9 for cleaning;

[0083] S5: Release horseradish peroxidase-streptavidin conjugate from enzyme storage chamber 10;

[0084] S6: Release the washing buffer from the third buffer chamber 11 for washing;

[0085] S7: Release the tetramethylbenzidine solution from the bottom chamber 12 and incubate at room temperature in the dark;

[0086] S8: Release the sulfuric acid solution in the termination chamber 13;

[0087] S9: After the reaction is completed, the solution in reaction detection chamber 3 is tested and analyzed.

[0088] To better illustrate the method of using the centrifugal microfluidic chip for allergen detection in this application, the following detailed description is provided in conjunction with specific embodiments.

[0089] S1: Since all reagents except whole blood are pre-loaded on the chip body 1 in the centrifugal microfluidic chip of the present invention, when using it, the whole blood sample is added to the serum chamber 602 of the serum separation structure 6, and then centrifugation is started to separate the whole blood. Blood cells enter the blood cell chamber 601, and serum remains in the serum chamber 602. Then the rotation speed is reduced, and the serum passes through the siphon valve 31 through the main channel 5 and the frustum-shaped channel 4 into the reaction detection chamber 3 containing polystyrene balls coated with anti-human IgE antibodies. Then, by changing the direction and amplitude of the chip body 1, the liquid is mixed and oscillated. At the same time, the anti-human IgE antibody coated on the polystyrene ball captures the specific IgE antibody, namely sIgE antibody, in the serum. Then, centrifugation is performed to discharge the mixture into the waste chamber 2 through the first burst valve 21.

[0090] S2: The washing buffer in the first buffer chamber 7 is released by centrifugation and flows through the second burst valve 22, main channel 5, and frustum-shaped channel 4 to the reaction detection chamber 3 for cleaning. The chip body 1 is oscillated back and forth. Then, the washing buffer is discharged into the waste chamber 2 through the first burst valve 21. This process is repeated three times based on the structure of the first buffer chamber 7. Cleaning three times can more thoroughly rinse the residual liquid after reagent hybridization in the reaction detection chamber 3, reduce false positives, and make the detection more accurate.

[0091] S3: The allergen-biotin in the allergen chamber 8 is released by changing the centrifugal speed. It passes through the third burst valve 23, the main channel 5, and the frustum-shaped channel 4 to the reaction detection chamber 3 for mixing and incubation. At the same time, the anti-human IgE antibody-sIgE antibody coated with polystyrene balls in the reaction detection chamber 3 will react with the allergen-biotin to form a complex of the three. Then, the liquid after the reaction is discharged into the waste chamber 2 through the first burst valve 21.

[0092] S4: The washing buffer in the second buffer chamber 9 is released through the fourth burst valve 22, the main channel 5, and the frustum-shaped channel 4 to the reaction detection chamber 3 for cleaning, and then discharged into the waste chamber 2. This process is repeated three times based on the structure of the second buffer chamber 9.

[0093] S5: Adjust the centrifugation speed to release the horseradish peroxidase-streptavidin conjugate solution in enzyme storage chamber 10. The solution passes through the fifth burst valve 25, main channel 5, and frustum-shaped channel 4 to the reaction detection chamber 3 for thorough mixing and incubation. At the same time, the biotin sites in the complex on the polystyrene spheres will react with the avidin sites in the horseradish peroxidase-streptavidin conjugate solution to form a complex of the four. The incubated liquid is discharged into waste chamber 2 through the first burst valve 21.

[0094] S6: The washing buffer in the third buffer chamber 11 is released through the sixth burst valve 26, the main channel 5, and the frustum-shaped channel 4 to the reaction detection chamber 3 for cleaning, and then discharged into the waste chamber 2. This process is repeated three times based on the structure of the third buffer chamber 11.

[0095] S7: Subsequently, the centrifugation speed of the chip body 1 is increased to release the tetramethylbenzidine solution in the bottom liquid chamber 12. This solution then passes through the seventh rupture valve 27, main channel 5, and frustum-shaped channel 4 to the reaction detection chamber 3 for a colorimetric reaction. After incubation with the tetramethylbenzidine solution, the reaction solution turns blue. Quantitative or qualitative analysis can usually be performed by measuring the absorbance at the corresponding wavelength or by visual observation. If the color is too dark, it indicates a high concentration of sIgE in the serum; if the color is too light or colorless, it indicates a low concentration of sIgE in the serum or the absence of sIgE corresponding to a certain allergen, meaning the individual is not allergic to that allergen.

[0096] S8: The sulfuric acid solution in the termination chamber 13 is released by changing the centrifugal speed of the chip body 1 through the eighth rupture valve 27, main channel 5, and frustum-shaped channel 4 to the reaction detection chamber 3 to terminate the reaction. The sulfuric acid solution can prevent the reaction between tetramethylbenzidine solution and horseradish peroxidase. The principle is: first, the sulfuric acid solution destroys the activity of horseradish peroxidase, causing the enzyme to lose its catalytic function; second, the pH decreases, turning the blue reaction solution into a yellow reaction solution. Because the reaction between tetramethylbenzidine solution and horseradish peroxidase continues, it will affect the experimental results, thus requiring the sulfuric acid solution to terminate the reaction and make the detection results more accurate.

[0097] S9: Subsequently, the absorbance of the solution in reaction chamber 3 was measured and analyzed using an ELISA reader in the dark. Absorbance detection utilizes an ELISA reader: the light emitted by the light source is converted into monochromatic light through a filter or monochromator. This monochromatic light enters the final reaction solution sample; part is absorbed by the sample, and the other part passes through the final reaction solution sample and illuminates the photodetector. The photodetector converts the light signal of the final reaction solution sample into a corresponding electrical signal. This electrical signal undergoes pre-amplification, logarithmic amplification, analog-to-digital conversion, and other signal processing before being sent to a microprocessor for data processing and calculation. Finally, the information is displayed on a monitor and printed. Absorbance detection results: The absorbance value is calculated by the ELISA reader, as shown below. I0 represents the light intensity of a substance before light absorption, and I represents the light intensity after light absorption. The concentration of sIgE in a patient sample is directly proportional to the absorbance value. The concentration of the patient's sIgE antibody can be determined by using a standard curve with the measured absorbance values. Please refer to [link to relevant documentation]. Figure 6 The patient's sIgE antibody concentration can be compared with the serum sIgE concentration grading standard to determine the level. Please refer to [link to relevant documentation]. Figure 7This allows for the assessment of the patient's allergic reaction. In summary, the process involves first using a microfluidic chip to detect the final yellow reaction solution; then, using an ELISA reader to determine the OD value (absorbance value); next, using a standard curve to determine the patient's sIgE antibody concentration; and finally, using a serum sIgE concentration grading system to determine the allergy level. Based on these test results, clinicians can use the patient's medical history, physical examination, and other clinical information, including in vivo tests, as an auxiliary means of diagnosing IgE-mediated allergic diseases.

[0098] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0099] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A centrifugal microfluidic chip for allergen detection, characterized in that, The chip includes a chip body and a plurality of microstructures disposed circumferentially on the chip body. The chip body has a rotation center, and the microstructures include: The waste chamber is located at the farthest end of the rotation center; A reaction detection chamber is provided in which a carrier is coated with anti-human IgE antibodies. One end of the reaction detection chamber is connected to the waste chamber through a first burst valve. A frustum-shaped channel, the narrow end of which is connected to the other end of the reaction detection chamber, and the wide end of which is connected to the main channel; A serum separation structure, comprising a blood cell chamber and a serum chamber connected to the blood cell chamber, wherein the serum chamber is connected to the main channel via a siphon valve; The first buffer chamber is connected to the main channel via at least one second burst valve; An allergen chamber, which is connected to the main channel via a third burst valve, is used to pre-load allergen-biotin; The second buffer chamber is connected to the main channel via at least one fourth burst valve; An enzyme storage chamber, which is connected to the main channel via a fifth burst valve, is used to pre-fill horseradish peroxidase-streptavidin conjugate solution; The third buffer chamber is connected to the main channel via at least one sixth burst valve; The bottom liquid chamber is connected to the main channel via a seventh rupture valve and is used to pre-fill tetramethylbenzidine solution. The termination liquid chamber is connected to the main channel via an eighth rupture valve and is used to pre-fill sulfuric acid solution.

2. The centrifugal microfluidic chip for allergen detection according to claim 1, characterized in that, The carrier is a polystyrene ball with a diameter of 115-150 μm.

3. The centrifugal microfluidic chip for allergen detection according to claim 1, characterized in that, A first microchannel connects one end of the reaction detection chamber to the waste chamber, the first rupture valve is connected to the first microchannel, and a second microchannel connects the narrow end of the frustum-shaped channel to the other end of the reaction detection chamber.

4. The centrifugal microfluidic chip for allergen detection according to claim 3, characterized in that, The width of the first microchannel and the width of the second microchannel are both 90-110μm.

5. The centrifugal microfluidic chip for allergen detection according to claim 1, characterized in that, The angle between the generatrix of the frustum-shaped channel and its center line is 20°-50°.

6. The centrifugal microfluidic chip for allergen detection according to claim 1, characterized in that, The width of the main channel is 500-800μm.

7. The centrifugal microfluidic chip for allergen detection according to claim 1, characterized in that, The blood cell chamber is circular, and / or the serum chamber is fan-shaped.

8. The centrifugal microfluidic chip for allergen detection according to claim 1, characterized in that, The diameters of the first, third, fifth, seventh, and eighth rupture valves are all 1.6-2.6 mm.

9. The centrifugal microfluidic chip for allergen detection according to claim 1, characterized in that, The diameters of the second, fourth, and sixth rupture valves are all 0.8-1.3 mm.

10. A method of using a centrifugal microfluidic chip for allergen detection, characterized in that, Using the centrifugal microfluidic chip as described in any one of claims 1-9 includes the following steps: (1) A whole blood sample is added to the serum separation structure, and the separated serum enters the reaction detection chamber; (2) Release the washing buffer in the first buffer chamber into the reaction detection chamber for cleaning; (3) Release the allergen-biotin in the allergen chamber into the reaction detection chamber; (4) Release the washing buffer in the second buffer chamber into the reaction detection chamber for cleaning; (5) Release the horseradish peroxidase-streptavidin conjugate solution in the enzyme storage chamber into the reaction detection chamber; (6) Release the washing buffer in the third buffer chamber into the reaction detection chamber for cleaning; (7) Release the tetramethylbenzidine solution in the bottom liquid chamber into the reaction detection chamber; (8) Release the sulfuric acid solution in the termination chamber into the reaction detection chamber; (9) After the reaction is completed, the solution in the reaction detection chamber is detected and analyzed.

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