CN-122017221-A - Centrifugal-lateral flow immune micro-fluidic chip for allergen and component sIgE combined detection

CN122017221ACN 122017221 ACN122017221 ACN 122017221ACN-122017221-A

Abstract

The invention is suitable for the technical field of in-vitro diagnosis, and provides a centrifugal-lateral flow immune microfluidic chip for combined detection of allergen and component sIgE, which comprises a chip body, wherein the chip body fuses a centrifugal microfluidic functional area and a lateral flow microfluidic functional area; according to the invention, an indirect immunoassay mode of 'firstly adding a sample, capturing an antibody to be detected by an antigen probe to form a complex-capturing the complex by an antigen probe, capturing by a microfluidic detection unit streptavidin-redissolving a labeled antibody-combining the labeled antibody with the antigen probe-the antibody to be detected' is adopted, and through accurate adaptation of a channel aperture and centrifugal force parameters, the reaction is ensured to be finished in a reaction unit before entering a lateral flow channel, at least 2 allergens/components can be synchronously detected, the sample consumption is less than or equal to 10 mu L, the detection time is less than or equal to 30 minutes, the sensitivity is improved by more than 10 times compared with that of the traditional method, and the method is suitable for basic layers, emergency and children screening scenes.

Inventors

YU YANG
YANG XIAOHUI
XUE SHAN
LI HUIQIANG
Li Daixian

Assignees

天津医科大学
普微(天津)生物技术有限公司

Dates

Publication Date: 20260512
Application Date: 20260210

Claims (10)

1. The centrifugal-lateral flow immune microfluidic chip for the combined detection of allergen and component sIgE is characterized by comprising a chip body, wherein the chip body fuses 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 distribution unit, a plurality of reaction units, a lateral flow detection unit and a waste liquid collection unit; The specimen loading area and the marker loading area are respectively communicated with the liquid distribution unit through liquid flow channels, the liquid distribution unit is communicated with the reaction units, the reaction units are communicated with the lateral flow detection units, and the lateral flow detection units are communicated with the waste liquid collection unit; The reaction unit is preloaded with biotinylated capture antigen, the marker loading region is preloaded with fluorescent 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 provided with a liquid flow control structure, the channel aperture of the chip body is accurately matched with centrifugal force parameters, liquid flow is driven by stage centrifugation, meanwhile, the capture reaction time is prolonged by means of capillary driving force, and when centrifugal force breaks through the explosion rotating speed of the valve in each stage, the method can be realized in sequence: The sample is distributed to each reaction unit in an equal volume and combined with the biotinylation capture antigen to form a complex, the complex is captured by the streptavidin of the lateral flow detection unit, the complex is enriched, the free components are separated, the labeled antibody is dissolved and flows through the detection unit in the same path, and the complex is combined with the captured complex in the detection unit; The chip realizes allergen and component sIgE detection through two-step immune reaction, wherein the two-step immune reaction is sequentially the combination reaction of sIgE antibody and biotinylation capture antigen in a sample and the secondary combination reaction of fluorescence labeled anti-human IgE antibody and the product of the combination reaction.
2. The centrifugal-lateral flow immune microfluidic chip for combined detection of allergen and component sIgE according to claim 1, wherein the chip body is of a wedge-shaped or fan-shaped structure, the specimen loading area, the marker loading area, the positioning hole and the liquid distribution unit are sequentially distributed outwards from the center of a circle, the liquid distribution unit is connected with at least two radially arranged detection units, and each detection unit is connected with one reaction unit and one lateral flow microfluidic unit in series.
3. The centrifugal-lateral flow immunoassay chip for combined detection of allergen and component sIgE according to claim 1, wherein the specimen loading area is a cylindrical groove body with a volume not less than 70 μl; the marker loading area is a cylindrical groove body, and the volume is not less than 120 mu L; The liquid distribution unit is an arc-shaped groove body, and the volume is not less than 130 mu L; Each reaction unit is a cylindrical cavity with the volume not less than 12 mu L; The waste liquid collecting unit is a cylindrical cavity with the volume not less than 50 mu L.
4. The centrifugal-lateral flow immunomicro-fluidic chip for combined detection of allergen and component sIgE according to claim 1, wherein the drying mode of the fluorescent-labeled anti-human IgE antibody is vacuum freeze drying, and the physical isolation of the sample from the sample loading area is realized by the design of an independent chamber in the dry state, and the sample can flow only after the sample is dissolved by adding distilled water or buffer solution.
5. The centrifugal-lateral flow immunomicrofluidic chip for combined detection of allergen and component ige according to claim 1, wherein the liquid flow channel comprises a channel 1 connecting the specimen loading zone/marker loading zone and the liquid distribution unit, a channel 2 connecting the liquid distribution unit and the reaction unit, a channel 3 connecting the reaction unit and the waste liquid collection unit; The channel 1 and the channel 2 are of a downhill type structure; the liquid flow control structure is a capillary blast valve and is respectively arranged at the inlets of the channel 1, the channel 2 and the channel 3.
6. The centrifugal-lateral flow immune microfluidic chip for combined detection of allergen and component sIgE according to claim 5, wherein the pore diameter of the channel 1 is 200-220 μm, the depth is 60-65 μm, the explosion speed of the corresponding capillary explosion valve is 750-850rpm, and the primary distribution flow of the labeled antibody after sample/dissolution is adapted; The aperture of the channel 2 is 150-160 mu m, the depth is 50-55 mu m, the explosion speed of the corresponding capillary explosion valve is 1400-1550rpm, and the flow of the sample/labeled antibody entering the reaction unit is adapted; The aperture of the channel 3 is 50-55 mu m, the depth is 30-32 mu m, the explosion rotating speed of the corresponding capillary explosion valve is 2400-2550rpm, and the flow of the reaction liquid from the reaction unit to the lateral flow detection unit is adapted.
7. The centrifugal-lateral flow immune microfluidic chip for combined detection of allergen and component sIgE according to claim 1, wherein the chip body is formed by enclosing a top layer and a bottom layer to form a cavity and a liquid flow channel, the material is PDMS or PMMA, and the surface is subjected to plasma hydrophilic treatment.
8. The centrifugal-lateral flow immune microfluidic chip for combined detection of allergens and components sIgE according to claim 1, wherein the chip body is a combinable unit, a plurality of the chip bodies can be spliced through positioning holes to form a multi-channel detection chip, and at least 30 allergens and components can be covered by single detection.
9. The centrifugal-lateral flow immune microfluidic chip for combined detection of allergen and component ige according to claim 1, wherein the two-step immune reaction is specifically: The first reaction is that sIgE antibody in the specimen is specifically combined with the pre-loaded biotinylation capture antigen in the reaction unit to form a biotinylation antigen-to-be-detected sIgE antibody complex, after centrifugation is started, the complex flows out of the reaction unit and enters a microfluidic channel in the lateral flow detection unit to finally reach a detection point; The second reaction is that after the dissolved fluorescent marked anti-human IgE antibody is distributed in equal volume by a liquid distribution unit, the dissolved fluorescent marked anti-human IgE antibody flows to a detection point along the same microfluidic channel path, and is specifically combined with the captured biotinylation antigen-to-be-detected sIgE antibody complex to form a biotinylation antigen-to-be-detected sIgE antibody-fluorescent marked anti-human IgE antibody ternary complex.
10. The centrifugal-lateral flow immune microfluidic chip for combined detection of allergen and component sIgE according to claim 1, wherein the technical flow and the dynamic mechanism of the lateral flow detection unit are as follows: firstly, a sample is distributed to each reaction unit in an equal volume through a liquid distribution unit, and is combined with a biotinylation capture antigen to form a compound, and the compound flows to a detection point through a microfluidic channel to realize primary targeted migration; Secondly, under the action of capillary driving force, detecting the specific combination of the pre-coated streptavidin and the biotin in the complex, realizing the rapid enrichment of ternary complex precursors, and simultaneously carrying out primary separation of unbound free components along a channel; thirdly, the labeled antibody flows through the microfluidic channel to reach a detection point through the same distribution path, and is combined with the enriched complex to form a ternary complex, and the remaining free fluorescent labeled antibody enters a waste liquid collecting unit under the assistance of subsequent centrifugation to complete thorough separation and washing; In the whole process, the capillary driving force provides sufficient reaction time for the specific combination of the complex and the streptavidin and the labeled antibody, ensures the detection specificity and sensitivity, and accords with the core technical principle of lateral flow immunoassay.

Description

Centrifugal-lateral flow immune micro-fluidic chip for allergen and component sIgE combined detection Technical Field The invention belongs to the technical field of in-vitro diagnosis, and particularly relates to a centrifugal-lateral flow immune microfluidic chip for combined detection of allergen and component sIgE. Background The current clinical detection core of allergen and component specific IgE (IgE) relies on immunoassay technology, and the mainstream methods include enzyme-linked immunosorbent assay (ELISA), light-emitting enzyme immunoassay (CLIA) and spot enzyme immunoassay (Dot-ELISA). The ELISA technology is mature, the reagent is easy to standardize, the method is a clinical basic detection means, the CLIA automation degree is high, the detection flux is large, the method is suitable for screening a large amount of samples, the Dot-ELISA operation is relatively simple and convenient, the cost is low, and the method has a certain application scene in basic medical institutions. The technologies are based on the specific binding principle of antigen and antibody, and the qualitative or quantitative detection of the target antibody is realized through signal amplification, so that an important basis is provided for diagnosis of allergic diseases. The prior art has the following defects: The multi-index joint detection capability is limited, the prior art is difficult to synchronously detect a plurality of allergens/components for a single time, the full spectrum allergy screening needs to detect a plurality of samples for a plurality of times, the detection combination cannot be dynamically adjusted according to the regional allergy epidemic characteristics or the individual risks, the serum utilization rate is low, the multi-sensitive patients need to sample for a plurality of times, and the sampling burden and the detection time are increased; the operation flow is complicated and the efficiency is low, the operation steps of the prior art are complex (such as ELISA needs 8-10 steps of incubation, washing and sample addition), the detection period is as long as 120-180 minutes, the dependence on professional equipment and operators is high, the manual operation error is difficult to control, and the requirements of fast-paced scenes such as rapid diagnosis of emergency anaphylactic shock, high-efficiency screening of pediatric outpatient service and the like can not be met; The detection sensitivity and the quantification accuracy are insufficient, the prior art lacks a high-efficiency signal amplification and compound enrichment mechanism, the detection lower limit is generally higher than 0.1kUA/L, the low-concentration sIgE antibody is easy to leak detection, the clinical leak diagnosis rate is higher, the quantification range is narrower, the variation Coefficient (CV) between batches is more than 5%, and the long-term curative effect monitoring requirement of early sensitization screening and allergy treatment of infants is difficult to be met; The adaptability of the trace sample is poor, the single detection in the prior art needs to consume 100-500 mu L of serum sample, the blood volume of children (especially infants) is limited, the risk is high and the compliance is poor when the blood is sampled for multiple times, the detection can not be completed in a trace blood sampling mode such as fingertip blood, heel blood and the like, and the popularity and the safety of allergy screening of the children are limited; the chip structure and the reaction control are complex, part of the multi-index detection chip depends on an open valve to control liquid flow, so that the chip structure is complex, the manufacturing cost is high, the problem of failure of liquid flow control is easy to occur, and the chip is not suitable for a basic layer low-cost application scene; therefore, a centrifugal-lateral flow immune microfluidic chip for combined detection of allergen and component sIgE is needed to solve the above problems. Disclosure of Invention The embodiment of the invention aims to provide a centrifugal-lateral flow immune micro-fluidic chip for combined detection of allergen and component sIgE, so as to solve the problems in the background art. In order to achieve the above purpose, the present invention provides the following technical solutions: The centrifugal-lateral flow immune microfluidic chip for the combined detection of allergen and component sIgE comprises a chip body, wherein the chip body fuses 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 distribution unit, a plurality of reaction units, a lateral flow detection unit and a waste liquid collection unit; The specimen loading area and the marker loading area are respectively communicated with the liquid distribution unit through liquid flow channels, the liquid distribution unit is communicated