Centrifugal-lateral flow immune micro-fluidic chip for combined detection of allergen and component sIgE

Abstract

The invention is applicable to the technical field of in-vitro diagnosis, and provides a centrifugal-lateral flow immune micro-fluidic chip for combined detection of allergens and a component sIgE, the centrifugal-lateral flow immune micro-fluidic chip comprises a chip body, and the chip body is fused with a centrifugal micro-fluidic functional area and a lateral flow micro-fluidic functional area; according to the method, an indirect immunoassay mode of firstly adding a specimen, capturing a to-be-detected antibody by an antigen probe to form a compound, capturing the compound by a microfluidic detection unit streptavidin, then dissolving a labeled antibody, combining the labeled antibody with the antigen probe and obtaining the to-be-detected antibody compound is adopted; the reaction is firstly completed in the reaction unit and then enters the lateral flow channel, at least two allergens / components can be synchronously detected, the sample dosage is less than or equal to 10 microliters, the detection time is less than or equal to 30 minutes, the sensitivity is improved by more than 10 times compared with that of a traditional method, and the method is adaptive to grassroots, emergency treatment and children screening scenes.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic technology, and particularly relates to a centrifugal-lateral flow immunomicrofluidic chip for the combined detection of allergens and their components sIgE. Background Technology

[0002] Currently, the core clinical detection of allergen and component-specific IgE (sIgE) relies on immunoassay techniques, with mainstream methods including enzyme-linked immunosorbent assay (ELISA), luminescent enzyme immunoassay (CLIA), and dot-ELISA. Among these, ELISA is a mature technology with easily standardized reagents, serving as a fundamental clinical detection method; CLIA boasts a high degree of automation and throughput, making it suitable for large-scale sample screening; and Dot-ELISA is relatively simple to operate and low-cost, finding some application in primary healthcare institutions. These technologies are all based on the principle of specific binding between antigens and antibodies, using signal amplification to achieve qualitative or quantitative detection of target antibodies, providing crucial evidence for the diagnosis of allergic diseases.

[0003] The existing technology has the following shortcomings: Limited multi-indicator combined detection capability: Existing technologies are difficult to detect multiple allergens / components simultaneously in a single test. Full spectrum allergy screening requires multiple tests and consumes multiple samples. Furthermore, it is impossible to dynamically adjust the test combination according to regional allergy prevalence characteristics or individual risk. Serum utilization is low, and patients with multiple allergies need to be sampled multiple times, which increases the sampling burden and testing time. The operation process is cumbersome and inefficient: Existing technologies have complicated operation steps (such as ELISA, which requires 8-10 steps of incubation, washing, and sample addition), and the detection cycle is as long as 120-180 minutes. They are highly dependent on professional equipment and operators, and human operation errors are difficult to control, which cannot meet the needs of fast-paced scenarios such as rapid diagnosis of emergency anaphylactic shock and efficient screening in pediatric outpatient clinics. Insufficient detection sensitivity and quantitative accuracy: Existing technologies lack efficient signal amplification and complex enrichment mechanisms, and the detection limit is generally higher than 0.1 kUA / L. Low concentrations of sIgE antibodies are prone to being missed, resulting in a high rate of missed diagnoses in clinical practice. At the same time, the quantitative range is narrow and the inter-batch coefficient of variation (CV) often exceeds 5%, making it difficult to meet the needs of early sensitization screening in infants and young children and long-term efficacy monitoring of allergy treatment. Poor adaptability of micro-samples: Current technology requires 100-500μL of serum sample for a single test, while children (especially infants) have limited blood volume. Multiple blood collections pose a high risk and have poor compliance. Testing cannot be completed through micro-blood collection methods such as finger prick blood or heel blood, which limits the accessibility and safety of allergy screening for children. Complex chip structure and reaction control: Some multi-index detection chips rely on open valves to control the liquid flow, resulting in complex chip structure, high manufacturing cost, and easy liquid flow control failure, making them unsuitable for low-cost application scenarios at the grassroots level; Therefore, centrifugal-lateral flow immunomicrofluidic chips for the combined detection of allergens and their components (sIgE) are needed to solve the above problems. Summary of the Invention

[0004] The purpose of this invention is to provide a centrifugation-lateral flow immunomicrofluidic chip for the combined detection of allergens and their components sIgE, in order to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A centrifugal-lateral flow immunomicrofluidic chip for the combined detection of allergens and their components using sIgE, comprising a chip body that integrates a centrifugal microfluidic functional area and a lateral flow microfluidic functional area; The chip body is provided with a specimen loading area, a marker loading area, a liquid dispensing unit, multiple reaction units, a lateral flow detection unit, and a waste liquid collection unit; The specimen loading area and the marker loading area are respectively connected to the liquid distribution unit through the liquid flow channel. The liquid distribution unit is connected to each of the reaction units. Each of the reaction units is connected to the lateral flow detection unit. The lateral flow detection unit is connected to the waste liquid collection unit. The reaction unit is preloaded with biotinylated capture antigen (antigen probe), the label loading region is preloaded with fluorescently labeled anti-human IgE antibody (solution powder) in a vacuum freeze-dried state, and the lateral flow detection unit is pre-coated with streptavidin. The liquid flow channel is equipped with a liquid flow control structure. The channel aperture of the chip body is precisely matched with the centrifugal force parameters. The liquid flow is driven by centrifugation in stages, and the capture reaction time is extended by the capillary driving force. When the centrifugal force in each stage exceeds the valve burst speed, the following can be achieved sequentially: The sample is allocated in equal volume to each reaction unit and binds to the biotinylated capture antigen to form a complex. The complex is captured by streptavidin in the lateral flow detection unit, thus achieving complex enrichment and separation of free components. After the labeled antibody is dissolved, it flows through the detection unit along the same path and binds to the complex already captured in the detection unit. The chip achieves the detection of allergens and components sIgE through a two-step immune reaction. The two-step immune reaction consists of the binding reaction of sIgE antibody in the sample with biotinylated capture antigen, and the secondary binding reaction of fluorescently labeled anti-human IgE antibody with the product of the above binding reaction.

[0006] Successfully addressing the core pain points of existing technologies such as low efficiency, cumbersome operation, high sample consumption, and insufficient sensitivity in multi-indicator detection, this technology achieves a technological breakthrough through four core designs: combining the advantages of rapid centrifugal reaction with efficient lateral flow enrichment and separation; labeling antibodies adaptable to all detection indicators; precise control of reaction and separation sequence; and support for channel expansion and micro-sample detection. It can simultaneously perform joint screening of at least 2 and up to 30 full-spectrum allergens, significantly reducing sample volume and detection time, and is suitable for multiple scenarios such as primary healthcare, emergency diagnosis, and early childhood screening.

[0007] In a further technical solution, the chip body has a wedge-shaped or fan-shaped structure, with the specimen loading area, the marker loading area, the positioning hole and the liquid distribution unit distributed sequentially from the center outwards. The liquid distribution unit is connected to at least two radially arranged detection units, and each detection unit is connected in series with a reaction unit and a lateral flow microfluidic unit. The wedge-shaped or fan-shaped structure adapts to the centrifuge rotation trajectory, ensuring uniform centrifugal force and providing a structural basis for multi-chip splicing; the radial arrangement avoids cross-contamination of different allergens / components, and combined with the flexible detection combination adjustment capability, it can support rapid screening of common allergens and meet the needs of full spectrum detection, significantly improving the flexibility and universality of detection solutions.

[0008] In a further technical solution, the specimen loading area is a cylindrical groove with a volume of not less than 70 μL; The marker loading area is a cylindrical groove with a volume of not less than 120 μL; The liquid distribution unit is an arc-shaped trough with a volume of not less than 130 μL; Each of the aforementioned reaction units is a cylindrical chamber with a volume of not less than 12 μL; The waste liquid collection unit is a cylindrical chamber with a volume of not less than 50 μL; The volume parameters are precisely matched around the miniaturized design, ensuring sufficient reaction space for small samples while also accommodating dissolving reagents and reaction waste liquids. The arc-shaped liquid distribution unit ensures that the sample and labeled antibody are evenly distributed in volume, ensuring consistent reagent concentration in each reaction unit. This effectively avoids incomplete reactions caused by volume issues and improves the accuracy and repeatability of multi-index detection results.

[0009] A further technical solution is that the fluorescently labeled anti-human IgE antibody is dried by vacuum freeze-drying. In the dried state, it is physically isolated from the sample loading area through an independent chamber design and can only flow after being dissolved in distilled water or buffer. Vacuum freeze-drying technology preserves antibody activity to the greatest extent, and the physical isolation of independent chambers prevents premature reaction between the sample and the labeled antibody from the source. The reaction sequence can be controlled without the need for additional physical valves, which greatly simplifies the chip structure, reduces manufacturing costs and processing difficulty, and the labeled antibody can be adapted to all detection indicators without the need for separate reagent design, which significantly improves the efficiency of multi-indicator detection.

[0010] A further technical solution is that the liquid flow channel includes a channel 1 connecting the specimen loading area / marker loading area and the liquid distribution unit, a channel 2 connecting the liquid distribution unit and the reaction unit, and a channel 3 connecting the reaction unit and the waste liquid collection unit; Both Channel 1 and Channel 2 are downslope structures; The fluid flow control structure is a capillary burst valve, which is respectively installed at the inlet of channel 1, channel 2 and channel 3; The downhill channel uses gravity to assist centrifugal force, reducing the risk of liquid stagnation; the capillary burst valve achieves precise timing control of liquid flow by balancing capillary force and centrifugal force, automatically advancing the detection process without external drive or manual intervention, perfectly meeting the two-step reaction requirements, greatly reducing human error, giving full play to the rapid response advantages of centrifugal microfluidics, and providing core support for shortening detection time.

[0011] In a further technical solution, the pore size of the channel 1 is 200-220μm and the depth is 60-65μm, corresponding to a capillary burst valve bursting speed of 750-850rpm, which is suitable for the initial distribution flow of samples / labeled antibodies after dissolution. The pore size of the channel 2 is 150-160μm and the depth is 50-55μm, corresponding to a capillary burst valve bursting speed of 1400-1550rpm, which is suitable for the flow of sample / labeled antibody into the reaction unit. The aperture of the channel 3 is 50-55μm and the depth is 30-32μm, corresponding to a capillary burst valve bursting speed of 2400-2550rpm, which is suitable for the flow of the reaction liquid from the reaction unit into the lateral flow detection unit. The gradient matching design of pore size and rotation speed forms a synergistic control of "low rotation speed distribution → medium rotation speed reaction → high rotation speed separation", which strictly ensures that the solution completes immune binding in the reaction unit first, and then enters the lateral flow channel for enrichment and separation. This effectively avoids detection deviations caused by insufficient reaction or incomplete separation, and significantly improves the accuracy and reliability of detection results.

[0012] In a further technical solution, the chip body is formed by a top layer and a bottom layer enclosing a chamber and a liquid flow channel, and the material is PDMS or PMMA, and the surface is treated with plasma hydrophilicity. The selected material has good biocompatibility, processability, and cost advantages, making it suitable for large-scale production; plasma hydrophilic treatment reduces the surface tension of the channel, avoids liquid adhesion and stagnation, and ensures smooth liquid flow and uniform distribution, especially suitable for the precise transmission of micro-samples, providing material guarantee for the stability and reliability of test results.

[0013] In a further technical solution, the chip body is a combinable unit, and multiple chip bodies can be spliced ​​together through positioning holes to form a multi-channel detection chip, which can cover at least 30 allergens and components in a single detection. The modular design breaks the limitations of single-chip detection indicators, enabling rapid screening of a small number of common allergens, and also meeting the needs of full-spectrum sensitization screening or epidemiological surveys through multi-chip splicing; the detection combination can be flexibly adjusted according to regional allergy characteristics and clinical testing purposes, significantly improving the universality and market adaptability of the technical solution.

[0014] A further technical solution is that the two-step immune response specifically comprises: The first reaction involves the specific binding of the sIgE antibody in the sample to the biotinylated capture antigen preloaded in the reaction unit, forming a "biotinylated antigen-sIgE antibody to be detected" complex. After centrifugation is initiated, this complex flows out of the reaction unit and into the microfluidic channel in the lateral flow detection unit, eventually reaching the detection point. Under the action of capillary driving force, the complex fully reacts with the streptavidin preloaded at the detection point and is specifically captured. The second reaction is that the dissolved fluorescently labeled anti-human IgE antibody is distributed in equal volume by the liquid distribution unit and flows to the detection point along the same microfluidic channel path, where it specifically binds to the captured "biotinylated antigen-detection sIgE antibody" complex to form a "biotinylated antigen-detection sIgE antibody-fluorescently labeled anti-human IgE antibody" ternary complex. The two-step immune response with specific binding design precisely targets the antibody, effectively avoiding interference from non-specific binding; the fluorescently labeled anti-human IgE antibody is compatible with the detection of all allergens / components, eliminating the need to design separate labeling reagents for different indicators, simplifying the reagent preloading process and detection operation, while laying a solid foundation for subsequent signal amplification and quantitative analysis.

[0015] A further technical solution, the technical process and power mechanism of the lateral flow detection unit are as follows: In the first step, the sample is dispensed into each reaction unit in equal volume via the liquid dispensing unit, where it binds to the biotinylated capture antigen to form a complex. The complex then flows to the detection point via the microfluidic channel, achieving initial targeted migration. In the second step, under the action of capillary driving force (lateral flow core dynamic characteristics), the streptavidin pre-coated at the detection point specifically binds to the biotin in the complex, realizing the rapid enrichment of the ternary complex precursor, while the unbound free components are initially separated along the channel. The third step involves the labeled antibody flowing through the microfluidic channel along the same distribution path to the detection point, where it binds to the enriched complex to form a ternary complex. The remaining free fluorescent labeled antibody is then collected in the waste liquid collection unit with the assistance of centrifugation to complete the thorough separation and washing. Throughout the process, the capillary driving force provides sufficient reaction time for the specific binding of the complex with streptavidin and the labeled antibody, ensuring the specificity and sensitivity of the detection, which is in line with the core technical principle of lateral flow immunoassay. The high affinity binding of streptavidin and biotin enables efficient enrichment of the complex, enhancing the detection signal intensity. The free label is completely separated from irrelevant components, significantly reducing background noise and greatly improving detection sensitivity and signal-to-noise ratio, resulting in a detection limit ≤0.1kUA / L, meeting the need for accurate detection of low-concentration sIgE antibodies in early sensitization of infants and young children.

[0016] Compared with the prior art, the beneficial effects of the present invention are: This invention enables simultaneous and flexible detection of multiple indicators: through a radial detection channel and a combinable design, a single test can simultaneously cover at least two allergens / components, and up to 30 allergens across the entire spectrum can be detected simultaneously; the liquid dispensing unit ensures equal volume distribution of samples, requiring only one serum sample to complete multidimensional screening, and the detection combination can be flexibly adjusted according to regional and seasonal allergy characteristics, solving the problems of low efficiency and poor sample utilization in existing technologies for multi-indicator detection, and reducing the number of samplings required for patients with multiple allergies; This invention is simple to operate and highly efficient in detection: the pre-loaded label drying design and antigen pre-load design eliminate multiple sample addition and washing steps; the combination of staged centrifugation and capillary burst valve enables orderly liquid flow without opening the valve, simplifying the operation steps to less than 3; the synergy of centrifugation and lateral flow microfluidics compresses the total detection time to ≤30 minutes (preferably 18-28 minutes), making it suitable for fast-paced scenarios such as emergency rooms and primary care clinics, and reducing human error; This invention offers high detection sensitivity and precise quantification: a signal amplification system is constructed by biotinylated antigen capture, fluorescently labeled antibody, and streptavidin, combined with the complex enrichment function of the lateral flow detection unit, achieving a detection limit ≤0.1kUA / L, with sensitivity more than 10 times higher than traditional ELISA; the high stability and uniform volume distribution of the fluorescent label ensure that the batch-to-batch CV is controlled at ≤4%, accurately capturing low concentrations of sIgE antibodies that cause early sensitization in infants and young children, meeting the needs of long-term efficacy monitoring; This invention offers excellent adaptability to micro-samples: the miniaturized chamber design allows for a single serum volume of ≤50μL, preferably ≤10μL (only 1 / 10-1 / 50 of traditional methods), enabling testing to be completed with micro-blood collection methods such as finger prick blood and heel prick blood; the small-volume design of the reaction unit ensures sufficient reaction with micro-samples, solving the problems of high sample consumption and difficulty in blood collection from children in existing technologies, and improving compliance and safety of allergy screening in children; This invention simplifies the chip structure and keeps costs under control: the "sample first, then antibody labeled" dry isolation design allows for control of the reaction sequence without opening valves, significantly reducing chip complexity and manufacturing costs, and meeting the low-cost application needs of primary healthcare institutions.

[0017] To more clearly illustrate the structural features and effects of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0018] Figure 1 This is a top view of the substrate (a) and top plate (b) structures of the present invention; Figure 2 This is a cross-sectional view of the present invention; Figure 3 This is a biomolecule distribution diagram of the present invention; Figure 4 This is a schematic diagram of the sample inflow path of the present invention; Figure 5 This is a schematic diagram illustrating the binding principle of the labeled antibody of the present invention; Figure 6 This is a schematic diagram of the allergen sIgE antibody detection results of the present invention. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0020] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0021] Example 1 like Figure 1-6As shown, this embodiment of the invention provides a centrifugation-lateral flow immunomicrofluidic chip for the combined detection of allergens and their components sIgE. The chip body comprises a wedge-shaped or fan-shaped PDMS structure, with a specimen loading area (6 mm in diameter, 2.5 mm in depth, and ≥70 μL in volume) arranged sequentially from the center outwards: a specimen tank (specimen loading area, 8 mm in diameter, 2.5 mm in depth, and ≥120 μL in volume), a positioning hole, and an arc-shaped liquid distribution transition tank (liquid distribution unit, 2.5 mm in depth, and ≥130 μL in volume). The liquid distribution transition tank connects to seven radial detection channels (lateral flow detection units), each channel connected in series with a reaction tank (reaction unit, 2.5 mm in diameter, 2.5 mm in depth, and ≥12 μL in volume). The end of each channel is connected to a waste liquid tank (waste liquid collection unit, 5 mm in diameter, 2.5 mm in depth, and ≥50 μL in volume). Both the liquid distribution transition tank and the waste liquid tank are equipped with vents.

[0022] In this embodiment, channel 1 is 4 mm long, 200 μm wide, and 60 μm deep; channel 2 is 5 mm long, 150 μm wide, and 50 μm deep; and channel 3 is 20 mm long, 50 μm wide, and 30 μm deep. The reaction chamber is preloaded with seven biotinylated allergens (antigen probes), including house dust mites, flour mites, and artemisia argyi. The distilled water tank is preloaded with vacuum-freeze-dried fluorescent microspheres labeled with anti-human IgE antibodies (solution powder). The capillary rupture valves correspond to centrifugation speeds of 800 rpm (first stage), 1500 rpm (second stage), and 2500 rpm (third stage). By matching the channel aperture with the rotation speed, the reaction is ensured. After being processed sequentially in the reaction chamber, the sample enters the lateral flow channel. During detection, 50 μL of serum sample is added to the sample tank. The liquid flow is driven by staged centrifugation to achieve equal volume distribution of the sample to each reaction unit. The sample binds to the biotinylated capture antigen to form a complex. The complex is captured by streptavidin in the microfluidic detection unit, achieving complex enrichment and separation of free components. 100 μL of distilled water is added to the distilled water tank to dissolve the labeled antibody. After dissolution, the labeled antibody flows through the detection unit along the same path and binds to the already captured complex in the reaction unit. The detection of 7 allergen sIgE is completed within 18 minutes, with a detection limit of 0.01 kUA / L.

[0023] Figure 2 Explanation of chip cross-section diagrams (A, B, C): Figure 2 In the diagram, A is a schematic diagram of the chip's top layer structure, showing the planar layout and functional opening design of the chip's top layer. The top layer is equipped with sample loading ports, distilled water loading ports, and venting ports corresponding to each detection channel. The sample loading ports are used for injecting samples and distilled water, and the venting ports are used to balance the air pressure inside and outside the chamber to prevent negative pressure stagnation during liquid flow. The top layer material is PDMS, which has undergone plasma hydrophilic treatment to ensure smooth liquid flow without sticking to the walls. Figure 2In the diagram, B is a schematic diagram of the chip's bottom layer structure, which shows the core functional chambers and microchannel layout of the chip's bottom layer. The bottom layer includes a sample tank, a distilled water tank, an arc-shaped liquid distribution unit, radially arranged reaction cells, microchannels, a capture detection zone, and a waste liquid tank. Biotinylated capture antigens are pre-loaded in the reaction cells, and streptavidin is pre-coated at the capture detection zone position. Each functional area is connected in series through microchannels of different pore sizes to form a complete liquid flow path.

[0024] Figure 2 In the diagram, C represents the overall structure of the chip. This diagram shows the cross-sectional structure after the top and bottom layers of the chip are bonded together, clearly demonstrating the sealed chambers and channels formed by the upper and lower layers. After the top layer covers the bottom layer, the sample tank and the distilled water tank form independent liquid storage spaces, and the microchannels form a sealed liquid flow path. Driven by centrifugal force, the liquid flows from the sample tank to the liquid distribution unit along the downhill microchannels, then enters the reaction tank, and finally flows into the waste liquid tank through the capture detection belt, completing the liquid flow transmission of the entire detection process.

[0025] Figure 3 Explanation of the biomolecular distribution diagrams (A, B) on the microarray: Figure 3 In the diagram, A represents the biomolecular distribution along the sample inflow path, illustrating the biomolecular binding process as the serum sample flows through the chip. After the sIgE antibody in the serum sample enters the reaction chamber, it specifically binds to the pre-loaded biotinylated allergen / component, forming a "biotinylated antigen-sIgE antibody" complex. This complex flows into the detection channel under centrifugal force and binds to the streptavidin pre-coated in the capture detection band, achieving targeted enrichment of the complex. Unbound free components continue to flow to the waste liquid pool.

[0026] Figure 3 In the diagram, B represents the biomolecular distribution along the distilled water inflow path, illustrating the biomolecular binding process as the labeled antibody dissolves and flows through the chip. After distilled water is added to the distilled water tank, it dissolves the pre-loaded vacuum freeze-dried fluorescent microspheres labeled with goat anti-human IgE antibody, forming a labeled antibody solution. This solution flows into the detection channel along the same path as the sample, binding with the "streptavidin-biotinylated antigen-sIgE antibody" complex already enriched in the capture detection band, forming a "streptavidin-biotinylated antigen-sIgE antibody-fluorescently labeled anti-human IgE antibody" ternary complex, ultimately generating a detectable fluorescent signal in the detection band.

[0027] Figure 4 Explanation of the Sample Inflow Path Diagram (AF): Figure 4 In the middle, A: The serum sample to be tested is added to the specimen tank through the sample addition hole. At this time, the capillary burst valve (capillary valve 1) at the outlet of the specimen tank is not broken, and the sample is restricted in the specimen tank and cannot enter channel 1. Figure 4 In the middle, B: Start the first stage of centrifugation (800 rpm). The centrifugal force overcomes the capillary resistance of capillary valve 1, and the sample is pushed into channel 1 and flows along the downhill channel to the arc-shaped liquid distribution unit, realizing the initial transfer of the sample. Figure 4 In the middle, C: After the liquid in the specimen tank is drained, the centrifuge speed is reduced to 0 rpm, and the sample is evenly distributed in the liquid distribution unit; at this time, the capillary valve 2 at the outlet of the liquid distribution unit is not broken, and the sample cannot enter the channel 2, which prepares for the subsequent equal volume distribution. Figure 4 In the middle, D: Start the second stage of centrifugation (1500 rpm). The centrifugal force overcomes the resistance of the capillary valve 2, and the sample is pushed into each radial channel 2 with an equal volume, and then flows into the corresponding reaction cell to achieve uniform distribution of samples in multiple detection channels. Figure 4 In the middle, E: After the liquid in the liquid distribution unit is emptied, the centrifuge speed is reduced to 0 rpm, and a 10-minute static incubation stage is entered; at this time, the capillary valve 3 at the outlet of the reaction cell is not breached, the sample is confined in the reaction cell, and the sIgE antibody and biotinylated antigen fully bind to form a complex. Figure 4 In the middle, F: Start the third stage of centrifugation (2500 rpm). The centrifugal force overcomes the resistance of capillary valve 3, and the complex in the reaction tank is pushed into channel 3 (detection channel). When the complex flows through the capture detection zone, it is captured by streptavidin, and the unbound free components are carried into the waste liquid tank by the centrifugal force, thus completing the enrichment and separation of the complex.

[0028] Example 2 The difference between this embodiment and Embodiment 1 is that: the chip body is made of PMMA and the surface is treated with plasma hydrophilicity; there are 10 detection channels and the liquid distribution transition groove is a ring structure; channel 1 is 4.5mm long, 220μm wide, and 65μm deep; channel 2 is 5.5mm long, 160μm wide, and 55μm deep; and channel 3 is 21mm long, 55μm wide, and 32μm deep.

[0029] In this embodiment, the reaction chamber is preloaded with 5 inhaled, 3 ingested, and 2 contact biotinylated allergens. The labeled antibody is a vacuum freeze-dried fluorescein-labeled anti-human IgE monoclonal antibody (solution powder). The centrifugation parameters are adjusted to 750 rpm for the first stage, 1400 rpm for the second stage, and 2400 rpm for the third stage, to adapt to the liquid flow characteristics of the PMMA material and the allocation requirements of 10 detection channels, ensuring that the reaction is fully completed in each reaction unit before entering the lateral flow detection unit. The chip can simultaneously detect 10 allergens, with a sample volume ≤10 μL, a detection time of 22 minutes, an inter-batch CV ≤4.5%, and a detection limit of 0.08 kUA / L, which is suitable for the multi-dimensional allergy screening needs of medium-sized medical institutions.

[0030] Example 3 The difference between this embodiment and embodiment 2 is that the chip body is a disk structure formed by splicing three wedge-shaped units, with a total of 30 detection channels; the labeled antibody is a quantum dot-labeled anti-human IgE antibody (fluorescence wavelength 500-850nm, vacuum freeze-dried), and the capture detection band of the lateral flow detection unit is pre-coated with streptavidin.

[0031] In this embodiment, three wedge-shaped or fan-shaped chip units are pre-loaded with inhaled, ingested, and contact allergens, respectively, and are spliced ​​together through positioning holes to achieve flexible expansion of detection indicators. The centrifugation parameters are adapted to the disc-shaped structure and set to 820 rpm for the first stage, 1550 rpm for the second stage, and 2550 rpm for the third stage. Through the precise matching of the three-stage channel aperture (220μm / 160μm / 55μm) and rotation speed, the consistency of liquid flow and controllable reaction sequence within each chip unit are ensured. During detection, a single collection of 20μL of finger prick blood can support the detection of 30 allergen combinations, and the detection is completed within 28 minutes. The detection limit is ≤0.1kUA / L, and the batch CV is ≤4%, which is suitable for early childhood full-spectrum sensitization screening and epidemiological survey scenarios.

[0032] Working principle and usage process of this invention: This chip utilizes centrifugal force to drive liquid flow, enabling an indirect two-step immune reaction and target antibody detection. The specific process is as follows: Sample allocation combined with the first immunization: Add the serum sample to be tested (≤50μL, preferably ≤10μL) to the sample loading area, fix the chip in the centrifuge through the positioning hole, and start the first stage of centrifugation (750-850rpm). The centrifugal force overcomes the resistance of the capillary burst valve at the inlet of channel 1 (pore size 200-220μm, depth 60-65μm), driving the sample into the arc-shaped liquid distribution unit along the downsloping channel. Start the second stage of centrifugation (1400-1550rpm), the centrifugal force overcomes the resistance of the capillary burst valve at the inlet of channel 2 (pore size 150-160μm, depth 50-55μm), and through the volume design of the distribution unit (≥130μL) adapted to the channel resistance, the sample is evenly distributed into each radially connected reaction unit (volume ≥12μL); Turn off the centrifuge and enter the static incubation stage (10 minutes). The sIgE antibody in the sample specifically binds to the biotinylated capture antigen preloaded in the reaction unit to form a "biotinylated antigen-sIgE antibody" complex, which lays the foundation for subsequent targeted capture. Complex migration and detection point capture: The third stage of centrifugation (2400-2550 rpm) is initiated. Centrifugal force overcomes the capillary burst valve resistance at the inlet of channel 3 (pore size 50-55 μm, depth 30-32 μm), driving the "biotinylated antigen-sIgE antibody" complex within the reaction unit to flow out along the channel. The complex flows through the microfluidic channel within the lateral flow detection unit, slowly passing through the detection point under the action of capillary driving force (the core dynamic characteristic of lateral flow). It fully reacts with the streptavidin pre-loaded at the detection point and is specifically captured. Unbound, irrelevant sample components are collected in the waste chamber to avoid cross-contamination. Labeled antibody dissolution and second immunization: Add distilled water or buffer (≥100μL) to the label loading area to dissolve the pre-loaded vacuum freeze-dried fluorescently labeled anti-human IgE antibody; maintain the centrifuge speed at 750-850rpm, and the centrifugal force overcomes the resistance of the capillary burst valve at the inlet of channel 1, driving the dissolved labeled antibody into the liquid distribution unit, and then distributing it in equal volumes, flowing to each detection point along the same microfluidic channel path; Under the action of capillary driving force, the fluorescently labeled anti-human IgE antibody specifically binds to the "biotinylated antigen-sIgE antibody" complex captured at the detection point, forming a ternary complex of "biotinylated antigen-sIgE antibody-fluorescently labeled anti-human IgE antibody". The remaining free fluorescently labeled antibody is collected into the waste liquid chamber, completing the core process of the two-step immune reaction. Signal detection and result output: Fluorescent markers enriched in the ternary complex at the detection site are excited using a fluorescence detection device, and the fluorescence signal intensity is collected. Based on a preset standard curve function, the sIgE antibody content corresponding to each allergen and component in the sample is calculated, and the test results are output in a semi-quantitative graded format (e.g., negative, weakly positive, positive, strongly positive) or quantitative numerical form. The entire testing process takes ≤30 minutes, requires no complex operations, and fully meets the rapid testing needs of various scenarios such as primary healthcare, emergency diagnosis, and early childhood screening.

[0033] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A centrifugal-lateral flow immunomicrofluidic chip for the combined detection of allergens and their components using sIgE, characterized in that, Includes a chip body, which integrates a centrifugal microfluidic functional area and a lateral flow microfluidic functional area; The chip body is provided with a specimen loading area, a marker loading area, a liquid dispensing unit, multiple reaction units, a lateral flow detection unit, and a waste liquid collection unit; The specimen loading area and the marker loading area are respectively connected to the liquid distribution unit through the liquid flow channel. The liquid distribution unit is connected to each of the reaction units. Each of the reaction units is connected to the lateral flow detection unit. The lateral flow detection unit is connected to the waste liquid collection unit. The reaction unit is preloaded with biotinylated capture antigen, the label loading region is preloaded with fluorescently labeled anti-human IgE antibody in a vacuum freeze-dried state, and the lateral flow detection unit is pre-coated with streptavidin. The liquid flow channel is equipped with a liquid flow control structure. The channel aperture of the chip body is precisely matched with the centrifugal force parameters. The liquid flow is driven by centrifugation in stages, and the capture reaction time is extended by the capillary driving force. When the centrifugal force in each stage exceeds the valve burst speed, the following can be achieved sequentially: The sample is allocated in equal volume to each reaction unit and binds to the biotinylated capture antigen to form a complex. The complex is captured by streptavidin in the lateral flow detection unit, thus achieving complex enrichment and separation of free components. After the labeled antibody is dissolved, it flows through the detection unit along the same path and binds to the complex already captured in the detection unit. The chip achieves the detection of allergens and components sIgE through a two-step immune reaction. The two-step immune reaction consists of the binding reaction of sIgE antibody in the sample with biotinylated capture antigen, and the secondary binding reaction of fluorescently labeled anti-human IgE antibody with the product of the above binding reaction.

2. The centrifugal-lateral flow immunomicrofluidic chip for combined detection of allergens and components sIgE according to claim 1, characterized in that, The chip body has a wedge-shaped or fan-shaped structure, with the sample loading area, the marker loading area, the positioning hole and the liquid distribution unit distributed outward from the center. The liquid distribution unit is connected to at least two radially arranged detection units, and each detection unit is connected in series with a reaction unit and a lateral flow microfluidic unit.

3. The centrifugal-lateral flow immunomicrofluidic chip for combined detection of allergens and components sIgE according to claim 1, characterized in that, The specimen loading area is a cylindrical groove with a volume of not less than 70 μL; The marker loading area is a cylindrical groove with a volume of not less than 120 μL; The liquid distribution unit is an arc-shaped trough with a volume of not less than 130 μL; Each of the aforementioned reaction units is a cylindrical chamber with a volume of not less than 12 μL; The waste liquid collection unit is a cylindrical chamber with a volume of not less than 50 μL.

4. The centrifugal-lateral flow immunomicrofluidic chip for combined detection of allergens and components sIgE according to claim 1, characterized in that, The fluorescently labeled anti-human IgE antibody is dried by vacuum freeze-drying. In the dried state, it is physically isolated from the sample loading area through an independent chamber design and can only flow after being dissolved in distilled water or buffer.

5. The centrifugal-lateral flow immunomicrofluidic chip for combined detection of allergens and their components using sIgE as described in claim 1, characterized in that, The liquid flow channel includes channel 1 connecting the specimen loading area / marker loading area and the liquid distribution unit, channel 2 connecting the liquid distribution unit and the reaction unit, and channel 3 connecting the reaction unit and the waste liquid collection unit; Both Channel 1 and Channel 2 are downslope structures; The fluid flow control structure is a capillary burst valve, which is respectively installed at the inlet of channel 1, channel 2 and channel 3.

6. The centrifugal-lateral flow immunomicrofluidic chip for combined detection of allergens and components sIgE according to claim 5, characterized in that, The pore size of channel 1 is 200-220μm and the depth is 60-65μm, corresponding to a capillary burst valve bursting speed of 750-850rpm, which is suitable for the initial distribution flow of sample / dissolved labeled antibody. The pore size of the channel 2 is 150-160μm and the depth is 50-55μm, corresponding to a capillary burst valve bursting speed of 1400-1550rpm, which is suitable for the flow of sample / labeled antibody into the reaction unit. The aperture of the channel 3 is 50-55μm and the depth is 30-32μm, corresponding to a capillary burst valve bursting speed of 2400-2550rpm, which is suitable for the flow of the reaction liquid from the reaction unit into the lateral flow detection unit.

7. The centrifugal-lateral flow immunomicrofluidic chip for combined detection of allergens and components sIgE according to claim 1, characterized in that, The chip body is formed by a top layer and a bottom layer enclosing a chamber and a liquid flow channel. The material is PDMS or PMMA, and the surface is treated with plasma hydrophilic treatment.

8. The centrifugal-lateral flow immunomicrofluidic chip for combined detection of allergens and components sIgE according to claim 1, characterized in that, The chip body is a combinable unit, and multiple chip bodies can be spliced ​​together through positioning holes to form a multi-channel detection chip, which can cover at least 30 allergens and components in a single detection.

9. The centrifugal-lateral flow immunomicrofluidic chip for combined detection of allergens and components sIgE according to claim 1, characterized in that, The two-step immune response is specifically as follows: The first reaction involves the specific binding of the sIgE antibody in the sample to the biotinylated capture antigen preloaded in the reaction unit, forming a "biotinylated antigen-sIgE antibody to be detected" complex. After centrifugation is initiated, this complex flows out of the reaction unit and into the microfluidic channel in the lateral flow detection unit, eventually reaching the detection point. Under the action of capillary driving force, the complex fully reacts with the streptavidin preloaded at the detection point and is specifically captured. The second reaction involves the dissolved fluorescently labeled anti-human IgE antibody being distributed in equal volume by the liquid distribution unit and flowing along the same microfluidic channel path to the detection point, where it specifically binds to the captured "biotinylated antigen-detection sIgE antibody" complex, forming a ternary complex of "biotinylated antigen-detection sIgE antibody-fluorescently labeled anti-human IgE antibody".

10. The centrifugal-lateral flow immunomicrofluidic chip for combined detection of allergens and their components using sIgE according to claim 1, characterized in that, The technical process and power mechanism of the lateral flow detection unit are as follows: In the first step, the sample is dispensed into each reaction unit in equal volume via the liquid dispensing unit, where it binds to the biotinylated capture antigen to form a complex. The complex then flows to the detection point via the microfluidic channel, achieving initial targeted migration. In the second step, under the action of capillary driving force, the streptavidin pre-coated at the detection point specifically binds to the biotin in the complex, achieving rapid enrichment of the ternary complex precursor, while the unbound free components are initially separated along the channel. The third step involves the labeled antibody flowing through the microfluidic channel along the same distribution path to the detection point, where it binds to the enriched complex to form a ternary complex. The remaining free fluorescent labeled antibody is then collected in the waste liquid collection unit with the assistance of centrifugation to complete the thorough separation and washing. Throughout the process, the capillary driving force provides sufficient reaction time for the specific binding of the complex with streptavidin and the labeled antibody, ensuring detection specificity and sensitivity, which is consistent with the core technical principle of lateral flow immunoassay.

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