Microfluidic chip for combined detection of hepatitis A / hepatitis E virus IgM antibodies and its application

Abstract

This invention proposes a microfluidic chip for the combined detection of hepatitis A / hepatitis E virus IgM antibodies and its application. The chip includes a substrate and a cover plate pressed onto the substrate. The substrate and cover plate enclose a microchannel, which is provided with a labeling area and a detection area. The labeling area includes a first labeling area on the lower surface of the cover plate and a second labeling area on the upper surface of the substrate. The detection area includes an HEV-IgM antibody detection area on the lower surface of the cover plate and a HAV-IgM antibody detection area on the upper surface of the substrate. An IgG antibody capture area is provided on the upper surface of the substrate directly below the sample injection well. The IgG antibody capture area contains magnetic microspheres containing anti-human IgG antibodies. The first labeling area contains labeled HEV antigen, the second labeling area contains labeled anti-human IgM antibody, the HEV-IgM antibody detection area contains anti-human IgM antibody, and the HAV-IgM antibody detection area contains HAV antigen. The capture method and the indirect method are used in parallel to achieve the combined detection of IgM antibodies related to the two viruses.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostics and immunoassay technology, and specifically relates to a microfluidic chip for the combined detection of hepatitis A / hepatitis E virus IgM antibodies and its application. Background Technology

[0002] Hepatitis A (HAA) and Hepatitis E (HEV) are acute viral hepatitis caused by different viruses (HAV and HEV), respectively, and are primarily transmitted through the fecal-oral route (e.g., contaminated water or food). Both types of hepatitis have a rapid onset, with common symptoms including fever, fatigue, loss of appetite, nausea, vomiting, abdominal pain, dark urine (like strong tea), and yellowing of the skin and sclera (jaundice). Generally, anyone experiencing unexplained acute hepatitis symptoms, especially those with weakened immune systems or those on long-term immunosuppressant therapy, should be tested for hepatitis A and E viruses to determine the cause. Furthermore, food handlers should be tested regularly to rule out current infection with hepatitis A / E viruses.

[0003] Laboratory diagnosis of acute hepatitis A / hepatitis E virus infection mainly includes molecular biology (nucleic acid detection) and immunological detection (antibody detection). Immunological or serological detection is a routine laboratory test method, with specific indicators being virus-specific IgM and IgG antibodies. IgM antibodies appear earlier and serve as a marker of acute viral infection; IgG antibodies, especially dynamic monitoring of IgG antibodies, show an increasing antibody titer index, which can also indicate the presence of current infection.

[0004] Generally, virus-associated IgM antibodies are detected using a capture method. This involves first coating a solid-phase material with mouse anti-human IgM antibodies to capture all IgM molecules in human serum. After washing to remove irrelevant substances, a specific antigen labeled with biotin (hepatitis A virus antigen or hepatitis E virus antigen) is added, and the signal is amplified and detected by streptavidin labeled with a tracer (enzyme).

[0005] HAV and HEV IgM antibodies are important serological diagnostic indicators for hepatitis A and hepatitis E virus infections. When using chemiluminescence analysis, they generally need to be detected separately. Colloidal gold immunochromatography, using an indirect mode, can achieve joint detection of HAV and HEV IgM antibodies, but non-specific binding often leads to false positive results. If a capture mode is used, separate detection is necessary to avoid mutual interference. Therefore, to achieve rapid joint detection of HAV and HEV IgM antibodies based on colloidal gold immunochromatography, the following issues need to be addressed:

[0006] (1) Indirect mode: In the indirect mode, the antigen is coated on the solid phase material. If there is a virus antigen-specific IgG antibody in the specimen, it will also bind to the antigen molecule, and the affinity is higher than that of the specific IgM antibody. As a result, the specific IgM antibody to be tested is not captured by the solid phase antigen, ultimately resulting in a false negative result (missed diagnosis).

[0007] (2) Capture mode: In the same NC membrane detection lane, the NC membrane is coated with mouse anti-human IgM antibody. The detection line (T) cannot distinguish between HAV-IgM antibody and HEV-IgM antibody. At the same time, the labeled antigen cannot be distinguished by the position of the detection line. In other words, the capture method based on colloidal gold (fluorescent) immunochromatography technology cannot achieve rapid detection of HAV and HEV IgM antibodies. Summary of the Invention

[0008] This invention aims to provide a microfluidic chip for the joint detection of hepatitis A / hepatitis E virus IgM antibodies and its application. By optimizing the chip design and detection method, it solves the problems existing in the prior art. Based on bidirectional microfluidic technology, it realizes the parallel detection of capture method and indirect method, and achieves the joint detection of IgM antibodies related to the two viruses.

[0009] To achieve the above objectives, the technical solution of the present invention is as follows: A microfluidic chip for the combined detection of hepatitis A / hepatitis E virus IgM antibodies includes a substrate and a cover plate pressed onto the substrate. The substrate and the cover plate enclose a microchannel with a height of 20-50 μm. The left end of the microchannel communicates with a buffer injection hole on the cover plate. A flow control valve is provided at the right end of the microchannel, controlling the opening and closing of the flow path of liquid in the microchannel to the flow control valve. From left to right, the microchannel is provided with a labeling area and a detection area. The labeling area includes a first labeling area on the lower surface of the cover plate and a second labeling area on the upper surface of the substrate. The detection area includes an HEV-IgM antibody detection area on the lower surface of the cover plate and a HAV-IgM antibody detection area on the upper surface of the substrate. The HEV-IgM antibody detection area and the HAV-IgM antibody detection area are arranged along the microchannel from left to right. It also includes a sample injection port, which is located between the detection area and the flow control valve. An IgG antibody capture area is provided on the upper surface of the substrate directly below the sample injection port. A magnetically controlled intercept valve is provided between the labeling area and the detection area of ​​the microchannel. The magnetically controlled intercept valve uses a magnet to attract and control the opening and closing of the flow path between the labeling area and the detection area in the microchannel. The IgG antibody capture area is coated with magnetic microspheres-anti-human IgG antibody. The first labeling area is coated with labeled HEV antigen. The second labeling area is coated with labeled anti-human IgM antibody. The HEV-IgM antibody detection area is coated with anti-human IgM antibody. The HAV-IgM antibody detection area is coated with HAV antigen.

[0010] In one embodiment of the present invention, the labeled anti-human IgM antibody is a labeled mouse anti-human IgM antibody, the anti-human IgM antibody is a mouse anti-human IgM antibody, and the HAV antigen is indirectly coated on the HAV-IgM antibody detection area by the mouse anti-human IgM antibody.

[0011] In one embodiment of the present invention, the first marking area and the second marking area overlap on a vertical plane.

[0012] In one embodiment of the present invention, the horizontal distance between the HEV-IgM antibody detection area and the buffer injection well is 30-35 mm, and the horizontal distance between the HEV-IgM antibody detection area and the buffer injection well is 40-45 mm.

[0013] In one embodiment of the present invention, a groove is provided on the lower surface of the cover sheet along its length direction, and the cover sheet forms a microchannel by means of the groove and the upper surface of the substrate, wherein the width of the microchannel is 2-3mm.

[0014] In one embodiment of the present invention, the labeled HEV antigen and labeled anti-human IgM antibody are fluorescent microspheres.

[0015] In one embodiment of the present invention, the cover plate is provided with a flow guide hole, the bottom of the magnetically controlled intercept valve passes through the flow guide hole and contacts the upper surface of the substrate, the top of the magnetically controlled intercept valve is provided with an iron material that can be attracted by a magnet, the bottom of the magnetically controlled intercept valve detaches from the flow guide hole after being attracted by magnetic force, and the bottom of the magnetically controlled intercept valve is provided with a water-absorbing material.

[0016] In one embodiment of the present invention, the flow control valve is a movable absorbent material, which moves to contact or move away from the microchannel.

[0017] On the other hand, the present invention also provides an application of the microfluidic chip in any of the above technical solutions for the combined detection of hepatitis A / hepatitis E virus IgM antibodies, comprising at least the following steps:

[0018] 1) Move the flow control valve to the far right to disconnect it from the microchannel. Apply a magnetic field to the substrate at the bottom of the sample injection well. Add diluted serum sample to the sample injection well to dissolve the magnetic microspheres (anti-human IgG antibodies) in the IgG antibody capture area. The sample liquid flows to the left and right. The liquid flowing to the right stops at the end of the microchannel due to surface tension, while the liquid flowing to the left can continue to flow. The lower liquid flows into contact with the upper surface of the substrate. When it reaches the HAV-IgM antibody detection area, the HAV-IgM antibody in the sample is captured, and the remaining irrelevant proteins are collected into the magnetically controlled intercept valve. The upper liquid flows into contact with the lower surface of the cover plate, and the IgM antibody in the sample is captured by the anti-human IgM antibody in the HAV-IgM antibody detection area. After all the liquid in the channel has been collected into the magnetically controlled intercept valve, remove the magnetically controlled intercept valve. At this time, the microchannel is restored to an open state.

[0019] 2) Move the flow control valve to the far left so that it is embedded in the microchannel. At the same time, remove the magnetic field at the sample injection hole. Add distilled water to the buffer injection hole to dissolve the buffer powder. Under the action of capillary driving force, it enters the microchannel and flows forward in a laminar flow state. When it moves to the labeling area, it dissolves the labeled HEV antigen in the first labeling area and the labeled anti-human IgM antibody in the second labeling area. Under the action of the second driving force on the right side of the microchannel, the liquid moves from left to right. In the top HEV-IgM antibody detection area, the labeled HEV antigen binds to the captured HEV-IgM antibody. In the bottom HAV-IgM antibody detection area, the labeled anti-human IgM antibody binds to the captured HAV-IgM antibody. The remaining substances are collected into the flow control valve. At the same time, the magnetic particles below the sample injection hole are also collected into the flow control valve.

[0020] 3) Read the signal values ​​of the HEV-IgM antibody detection area and the HAV-IgM antibody detection area using conventional microfluidics.

[0021] The microfluidic chip for combined detection of hepatitis A / hepatitis E virus IgM antibodies obtained through the above technical solution has the following beneficial effects:

[0022] 1. Because magnetic microspheres coated with anti-human IgG antibodies are used at the sample well, IgG molecules in the sample are bound and will not infiltrate the microfluidic channel under the action of the magnetic field, thus effectively avoiding the interference of IgG molecules, especially when detecting HAV-IgM by indirect method.

[0023] 2. Based on the laminar flow characteristics of the liquid in the microfluidic channel, HEV-IgM detection points are designed on the top layer and HAV-IgM detection points are designed on the bottom layer, which effectively avoids the mutual interference between the labeled molecules of the two detection systems, thereby realizing the joint detection of dual indicators.

[0024] 3. Because HAV antigens are difficult to purify or recombinantly express, indirect coating and indirect analysis modes are required. HEV antigens, on the other hand, are easily prepared and labeled, allowing for the use of antigen-labeled fluorescent microspheres, thus enabling a capture mode. The parallel use of these two analysis modes, distributed at the top and bottom layers respectively, constitutes independent detection systems, achieving joint detection of both indicators.

[0025] 4. Since the mouse anti-human IgM antibody captured by the capture method cannot distinguish the specificity of IgM, an indirect method for HAV-IgM is first performed, allowing the HAV antigen to capture the HAV-IgM antibody first, followed by the HEV-IgM capture method. That is, the HAV-IgM detection point is placed on the right side and contacts the sample first, while the HEV-IgM detection point is placed on the left side and contacts the sample later. This design can ensure that both indicators obtain satisfactory sensitivity. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the structure of the microfluidic chip (transparent) for the combined detection of hepatitis A / hepatitis E virus IgM antibodies described in this invention;

[0027] Figure 2 This is a schematic diagram of the cover plate (transparent) of the present invention;

[0028] Figure 3 This is a schematic diagram of the structure of the substrate described in this invention;

[0029] Figure 4 This is a cross-sectional view of the microfluidic chip for the combined detection of hepatitis A / hepatitis E virus IgM antibodies as described in this invention.

[0030] Figure 5This is a schematic diagram illustrating the principle of adding a sample to be tested using the layered microfluidic chip described in this invention.

[0031] Figure 6 This is a schematic diagram of the principle of adding buffer solution to the layered microfluidic chip described in this invention;

[0032] Figure 7 This is a biomolecular distribution diagram of the microfluidic chip for the combined detection of hepatitis A / hepatitis E virus IgM antibodies as described in this invention;

[0033] Figure 8 This invention is in Figure 7 Schematic diagram of the principle of adding the sample to be tested;

[0034] Figure 9 This invention is in Figure 8 A schematic diagram of the reaction stage of the sample under test on the chip;

[0035] Figure 10 This invention is in Figure 9 A schematic diagram showing how a sample to be tested on a chip adsorbs waste liquid through a magnetically controlled interception valve.

[0036] Figure 11 This invention is in Figure 10 A schematic diagram of the principle of adding buffer solution onto the chip;

[0037] Figure 12 This invention is in Figure 11 A schematic diagram of the process of marking the material binding stage on a chip.

[0038] In the figure, 1 is the substrate; 2 is the cover plate; 3 is the microchannel; 4 is the buffer injection well; 5 is the flow control valve; 6 is the sample injection well; 7 is the magnetically controlled intercept valve; 31 is the labeling area; 31a is the first labeling area; 31b is the second labeling area; 32 is the detection area; 32a is the HEV-IgM antibody detection area; and 32b is the HEV-IgM antibody detection area. Detailed Implementation

[0039] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0040] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0041] This invention relates to the field of immunoassay technology, specifically to a microfluidic chip for the combined detection of hepatitis A / hepatitis E virus IgM antibodies. Based on the laminar flow principle of lateral flow microfluidics and combining the characteristics of hepatitis A and hepatitis E virus antigens, the HEV-IgM antibody detection is placed on the top layer in a capture mode, while the HAV-IgM antibody detection is placed on the bottom layer in an indirect mode.

[0042] The present invention will be further explained and described below with reference to the embodiments and accompanying drawings. It should be understood that the present invention is not limited to the specific embodiments described.

[0043] like Figure 1-4 As shown, this invention proposes a microfluidic chip for the combined detection of hepatitis A / hepatitis E virus IgM antibodies, comprising a substrate 1 and a cover plate 2 pressed onto the substrate 1. The substrate 1 and the cover plate 2 enclose a microchannel 3, the height of which is 20-50 mm. μm, the left end of the microchannel 3 is connected to the buffer injection hole 4 on the cover plate 1, and the right end of the microchannel 3 is provided with a flow control valve 5. The flow control valve 5 controls the opening and closing of the flow path of liquid in the microchannel 3 to the flow control valve 5. The microchannel 3 is provided with a labeling area 31 and a detection area 32 from left to right. The labeling area 31 includes a first labeling area 31a on the lower surface of the cover plate 2 and a second labeling area 31b on the upper surface of the substrate 1. The detection area 32 includes an HEV-IgM antibody detection area 32a on the lower surface of the cover plate 2 and a HAV-IgM antibody detection area 32b on the upper surface of the substrate 1. The HEV-IgM antibody detection area 32a and the HAV-IgM antibody detection area 32b are arranged from left to right along the microchannel 3. The cover plate The substrate 1 is also provided with a sample injection port 6, which is located between the detection area 32 and the flow control valve 5. An IgG antibody capture area 33 is provided on the upper surface of the substrate 1 directly below the sample injection port 6. A magnetically controlled intercept valve 7 is provided between the labeling area 31 and the detection area 32 of the microchannel. The magnetically controlled intercept valve 7 uses a magnet to attract and control the opening and closing of the flow path between the labeling area 31 and the detection area 32 in the microchannel 3. The IgG antibody capture area 33 is coated with magnetic microspheres-anti-human IgG antibody. The first labeling area 31a is coated with labeled HEV antigen. The second labeling area 31b is coated with labeled anti-human IgM antibody. The HEV-IgM antibody detection area 32a is coated with anti-human IgM antibody. The HEV-IgM antibody detection area 32b is coated with HAV antigen.

[0044] In fluid mechanics, the Reynolds number (Re) is a key parameter for determining the flow state of a liquid. When Re is below a critical value, the flow is laminar. In microfluidic channels, Re < 100 can be considered laminar flow.

[0045] According to the formula for calculating the Reynolds number: Re = (ρ is the fluid density, v is the average flow velocity, Dh is the hydraulic diameter, and μ is the fluid dynamic viscosity). For a rectangular microchannel, Dh = (w is the channel width, h is the channel height). Since the width of the microchannel is much greater than its height, Dh can be simplified to Dh = 2h. Therefore, the height of the microchannel is the key parameter that determines the liquid flow state in the microchannel.

[0046] For common fluids (such as water and buffer solutions), at typical flow rates (0.1-10 mm / s), the channel height h is usually set to 10-100 μm to ensure Re < 100. However, for other liquids with faster flow rates, if the microchannel height is not designed properly, the internal flow will exhibit an irregular turbulent state, causing reactions at different levels to interfere with each other. Therefore, limiting the microchannel height to 20-50 μm ensures that any liquid flow exhibits a stable laminar flow state within the microchannel.

[0047] Therefore, the height of the microchannel in this application is limited to 20-50 μm, which can maintain the liquid flow in the microchannel in a stable laminar flow state, ensuring that the reactions of the HEV-IgM antibody detection layer and the HAV-IgM antibody detection layer proceed independently without interference.

[0048] The workflow diagram of the layered microfluidic chip is as follows:

[0049] A. Detection molecule capture stage: such as Figure 5 As shown, the flow control valve is switched to the far right, disconnecting it from the microchannel, and a magnetic field is applied to the substrate at the bottom of the sample injection well. After adding the sample to be tested into the sample injection well, the sample buffer and magnetic microspheres (pre-coated with anti-human-IgG antibody) are dissolved. The IgG in the sample is captured and fixed in its original position below the sample injection well under the magnetic field. The liquid flows to the left and right. Since the microchannel is not connected to the flow control valve, the liquid flowing to the right remains at the end of the microchannel due to the surface tension at the end and will not flow out spontaneously, while the liquid flowing to the left can continue to flow. When it reaches the detection area, the analyte binds to the solidified (coated) antigen / antibody in the detection area. When the liquid continues to flow and reaches the magnetically controlled intercept valve, it is intercepted. Since the water absorption force provided by the magnetically controlled intercept valve is greater than the capillary force in the microchannel, the liquid is immediately collected into the magnetically controlled intercept valve and will not continue to flow to the left. Once all the liquid flow in the microchannel has been collected into the magnetically controlled intercept valve, the magnetically controlled intercept valve is removed, and the microchannel is restored to unobstructed flow, ensuring that subsequent reagent flow and reactions are not disturbed.

[0050] B. The stage of marker substance binding: such as Figure 6As shown, the flow control valve is moved to the far left, embedding it within the microchannel, while the magnetic field is removed. Buffer solution is added to the buffer injection well; driven by capillary force, it enters the microchannel and dissolves the labeled material as it flows through the labeled area. The labeled material, carried by the flow, reaches the detection area and is captured by the corresponding substance there; the remaining flow is collected into the flow control valve. Simultaneously, magnetic particles at the sample well are also collected into the flow control valve.

[0051] The labeled anti-human IgM antibody is a mouse anti-human IgM antibody, and the anti-human IgM antibody is a labeled mouse anti-human IgM antibody. The HAV antigen is indirectly coated on the HAV-IgM antibody detection region 32b by the mouse anti-human IgM antibody.

[0052] The first marking area 31a and the second marking area 31b overlap on the vertical plane.

[0053] The horizontal distance between the HEV-IgM antibody detection area 32a and the buffer injection well 4 is 30-35 mm, and the horizontal distance between the HEV-IgM antibody detection area 32b and the buffer injection well 4 is 40-45 mm.

[0054] The cover plate 2 has a groove along its length on its lower surface. The cover plate 2 and the upper surface of the substrate 1 are enclosed by the groove to form a microchannel 3. The width of the microchannel 3 is 2-3 mm.

[0055] The labeled HEV antigen and labeled anti-human IgM antibody are labeled with fluorescent microspheres.

[0056] The cover plate 2 is provided with a flow guide hole. The bottom of the magnetically controlled intercept valve 7 passes through the flow guide hole and contacts the upper surface of the substrate 1. The top of the magnetically controlled intercept valve 7 is provided with an iron material that can be attracted by a magnet. After the magnetically controlled intercept valve 7 is attracted by magnetic force, the bottom of the magnetically controlled intercept valve 7 is separated from the flow guide hole. The bottom of the magnetically controlled intercept valve 7 is provided with a water-absorbing material.

[0057] The flow control valve 5 is made of a movable absorbent material, which moves to contact or move away from the microchannel.

[0058] Biomolecular distribution of a microfluidic chip for combined detection of hepatitis A / hepatitis E virus IgM antibodies, such as... Figure 7 As shown,

[0059] Combining the characteristics of hepatitis A virus antigen and hepatitis E virus antigen, the HEV-IgM antibody detection is set on the lower surface of the coverslip (top layer of the chip) using a capture mode; the HAV-IgM antibody detection is set on the upper surface of the substrate (bottom layer of the chip) using an indirect mode. The specific design is as follows:

[0060] (1) Indirect method for detecting HAV-IgM antibody

[0061] At the bottom layer of the chip, streptavidin is coated, followed by biotinylated anti-hepatitis A virus antibody (IgG), and then hepatitis A virus particles (antigen). Since hepatitis A virus antigens are mostly conformational epitopes and cannot be recombinantly expressed or isolated and purified, an indirect coating mode of antibody-hepatitis A virus particles is generally used. Simultaneously, dried fluorescent microspheres labeled with anti-human IgM antibody are placed in the microfluidic labeling area.

[0062] (2) HEV-IgM antibody detection by capture method

[0063] The top layer of the chip is coated with streptavidin and then combined with biotinylated mouse anti-human IgM antibody; at the same time, dried fluorescent microspheres labeled with recombinant hepatitis E virus recombinant antigen are placed in the microfluidic labeling area.

[0064] (3) Design to remove IgG interference

[0065] Place dried anti-human IgG coated magnetic microparticles (1200 nm in diameter) at the bottom of the sample well.

[0066] Since mouse anti-human IgM antibodies cannot distinguish the specificity of IgM, an indirect method for HAV-IgM is required first, allowing the HAV antigen to capture the HAV-IgM antibody before proceeding with the HEV-IgM capture method.

[0067] The detection principle is as follows:

[0068] 1) Detection molecule capture stage:

[0069] like Figure 8 As shown, the flow control valve is switched to the far right, disconnecting it from the microchannel, and a magnetic field is applied to the substrate at the bottom of the sample injection well to attract magnetic particles. 35 μL of diluted serum sample (1:100 dilution) is added to the sample injection well, dissolving the magnetic microspheres (pre-coated with anti-human IgG antibody) at the bottom of the well. At this point, IgG in the bottom flow is captured, while IgM antibody and serum protein flow to the left and right sides with the flow. Figure 9As shown, since the microchannel is not connected to the flow control valve at this time, under the action of the surface tension at the end, the liquid flowing to the right remains at the end of the microchannel and will not flow out spontaneously, while the liquid flowing to the left can continue to flow. During this process, the magnetic particles are fixed at their original positions at the bottom of the sample well under the magnetic field. During the liquid flow, the lower liquid flow contacts the inner surface of the bottom layer of the chip. When it reaches the HAV-IgM antibody detection area, the HAV-IgM antibody contained in the sample is captured by the HAV antigen immobilized here (if the sample contains HAV-IgG antibody, it will not affect the reaction of the HAV-IgM antibody detection area because it has been captured by the anti-human IgG antibody at the bottom of the sample well). The remaining irrelevant proteins are collected into the magnetically controlled intercept valve; the upper liquid flow contacts the inner surface of the top layer of the chip, and the IgM antibody contained in the sample is captured by the mouse anti-human IgM antibody in the HEV-IgM antibody detection area, such as Figure 10 As shown, after all the liquid flow in the channel has been collected into the magnetically controlled intercept valve, the intercept valve is removed, and the microchannel is restored to its unobstructed state.

[0070] 2) The binding stage of labeled substances:

[0071] like Figure 11 As shown, move the flow control valve to the far left, embedding it into the microchannel, and simultaneously remove the magnetic field from the sample loading well. Add distilled water to the buffer injection well to dissolve the buffer powder. Driven by capillary force, the powder enters the microchannel and flows forward in a laminar flow state, as shown. Figure 12 As shown, upon moving to the labeling area, the HEV antigen marker at the top layer and the mouse anti-human IgM antibody marker at the bottom layer dissolve, respectively. The liquid moves from left to right within the microchannel under the influence of the second driving force on the right side. In the top HEV-IgM antibody detection area, the HEV antigen marker binds to the captured HEV-IgM antibody; in the bottom HAV-IgM antibody detection area, the mouse anti-human IgM antibody marker binds to the captured HAV-IgM antibody. The remaining substances are collected into the flow control valve. Simultaneously, the magnetic particles at the lower end of the sample injection well are also collected into the flow control valve.

[0072] 3) Initiate the testing process. If a signal appears in the HAV-IgM antibody detection area at the bottom of the chip, it indicates a positive HAV-IgM antibody test; if a signal appears in the HEV-IgM antibody detection area at the top of the chip, it indicates a positive HEV-IgM antibody test. It should be noted that HAV and HEV co-infection is generally unlikely, and signals will not appear in both detection areas simultaneously.

[0073] Example

[0074] 1. Pre-processing of biological raw materials

[0075] (1) Coupling of magnetic microparticles with anti-human IgG antibodies

[0076] Following standard conjugation procedures: First, the magnetic microparticles (1200 nm in diameter) were washed and resuspended. Then, the carboxyl groups on the surface of the magnetic microparticles were activated using EDC and NHS. After full activation, the supernatant was removed by magnetic separation. Subsequently, the activated magnetic microparticles were mixed with anti-human IgG antibody and incubated. After the conjugation reaction was completed, the remaining active sites on the surface of the microparticles were blocked. The microparticles were then washed and resuspended with preservation solution (final concentration 10 mg / mL, magnetic microparticle count 2.62 × 10¹⁰ / mL) and stored at 4°C for later use.

[0077] (2) Fluorescent microsphere coupling procedure

[0078] Following the standard conjugation procedure: First, the fluorescent microspheres are washed and resuspended. Then, the carboxyl groups on the surface of the fluorescent microspheres are activated using EDC and NHS. Mouse anti-human IgM antibody is then added to the resuspended activated product, and the mixture is incubated at room temperature with shaking for 2 hours. After the conjugation reaction is complete, 2% BSA is added to block unreacted sites, and the reaction is terminated with sodium citrate. After centrifugation and washing, the microspheres are resuspended in storage solution and stored at 4°C for later use. The conjugation procedure for HEV recombinant antigen and fluorescent microspheres is similar.

[0079] (3) Biotin labeling procedure

[0080] First, 10 mM biotin solution was added to mouse anti-human IgM antibody solution (molar ratio 20:1), and the mixture was incubated on ice for 2 h. After the reaction, the reaction mixture was placed in a dialysis vessel and dialyzed overnight in PBS to remove free biotin molecules. Finally, the concentration of biotinylated capture antibody was determined using a UV spectrophotometer and stored at 4°C for later use. The biotin labeling method for anti-HAV antibodies is similar.

[0081] 2. Chip fabrication

[0082] Both the substrate and the coverslip were made of PMMA material. The microchannel structure, sample injection port, buffer injection port, drainage port, and other structures of the coverslip were designed using CAD software, and then the PMMA surface was processed using a CO2 laser etching machine.

[0083] 3. Biomolecular coating program on the top layer of the chip

[0084] First, 1.5 μL of streptavidin was spotted on the top layer of the chip corresponding to the HEV-IgM antibody detection area. After incubation for 1 h and washing, the chip was dried and then 1.6 μL of biotinylated mouse anti-human IgM antibody was spotted on the same detection area. Incubation was continued for another 1 h. After washing again, 1.2 μL of fluorescent microspheres conjugated with HEV antigen was spotted on the labeled area and dried at 37°C.

[0085] 4. Biomolecular coating program at the chip's underlying layer

[0086] First, 1.5 μL of streptavidin was spotted at the HAV-IgM antibody detection area on the bottom layer of the chip, incubated for 1 h, washed, and dried. Then, 1.6 μL of anti-HAV antibody was spotted at the same detection area, incubated for another 1 h, and washed. Next, 1.8 μL of HAV virus particles was spotted, and incubation continued for 1 h. After another wash, 1.2 μL of fluorescent microspheres conjugated with anti-human IgM antibody was spotted at the labeled area, and simultaneously, 1 μL of magnetic microparticles conjugated with anti-human IgG antibody was spotted at the sample well. The mixture was then dried at 37°C.

[0087] 5. Microfluidic chip assembly procedure

[0088] First, the magnetically controlled interceptor valve is embedded in the liquid guide hole of the top layer of the chip; second, the liquid flow control valve is placed in the waste liquid chamber of the top layer of the chip; finally, the top layer and the bottom layer of the chip are tightly bonded together.

[0089] 6. Testing Procedure

[0090] Three serum samples, S1, S2, and S3, were selected and subjected to the following tests:

[0091] First, turn the flow control valve to the far right to disconnect it from the microchannel, and apply a magnetic field to the bottom of the sample well. Add 35 μL of diluted serum sample (1:100 dilution) to the sample injection well to dissolve the magnetic microspheres coupled with anti-human IgG antibody at the bottom of the well. At this point, IgG in the bottom flow is captured, while IgM antibody and serum protein flow to the left and right. Since the microchannel is not connected to the flow control valve, the liquid flowing to the right remains at the end of the microchannel due to surface tension and will not flow out spontaneously, while the liquid flowing to the left continues to flow. During this process, the magnetic particles are fixed in their original position at the bottom of the sample well under the magnetic field. During the liquid flow, the lower liquid flow contacts the inner surface of the bottom layer of the chip. When it reaches the HAV-IgM antibody detection area, the HAV-IgM antibody in the sample is captured by the HAV antigen immobilized here, and the remaining irrelevant proteins are collected into the magnetically controlled intercept valve. The upper liquid flow contacts the inner surface of the top layer of the chip, and the IgM antibody in the sample is captured by the mouse anti-human IgM antibody in the HEV-IgM antibody detection area. After all the liquid flow in the channel has been collected into the magnetically controlled intercept valve, the intercept valve is removed, and the microchannel is restored to an unobstructed state.

[0092] Move the flow control valve to the far left, embedding it within the microchannel, and simultaneously remove the magnetic field from the sample loading well. Add distilled water to the buffer injection well to dissolve the buffer powder. Driven by capillary force, the powder enters the microchannel and flows forward in laminar flow. Upon reaching the labeling area, it dissolves the HEV antigen marker at the top layer and the mouse anti-human IgM antibody marker at the bottom layer. The liquid moves from left to right within the microchannel under the influence of a second driving force on the right side. At the HEV-IgM detection point at the top layer, the HEV antigen marker binds to the captured HEV-IgM antibody; at the HAV-IgM detection point at the bottom layer, the mouse anti-human IgM antibody marker binds to the captured HAV-IgM antibody. The remaining substances are collected into the flow control valve. Simultaneously, the magnetic particles at the sample loading well are also collected into the flow control valve.

[0093] 7. Experimental Results

[0094] Table 1. Experimental results of HAV-IgM and HEV-IgM antibody detection

[0095]

[0096] The above technical solutions only embody the preferred technical solutions of the present invention. Any modifications that may be made by those skilled in the art to certain parts thereof embody the principles of the present invention and fall within the protection scope of the present invention.

[0097] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0098] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicating orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the invention. Furthermore, the terms "first," "second," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined with "first," "second," etc., may explicitly or implicitly include one or more of that feature. In the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0099] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "setting" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal communication between two components. Those skilled in the art will understand the specific meaning of the above terms in this invention based on the specific circumstances.

Claims

1. A microfluidic chip for combined detection of IgM antibodies against hepatitis A and E viruses, comprising a substrate and a cover plate pressed on the substrate, wherein the substrate and the cover plate enclose a microchannel, the height of the microchannel is 20-50 μm, the left end of the microchannel is in communication with a buffer injection hole formed on the cover plate, the right end of the microchannel is provided with a liquid flow control valve, the liquid flow control valve controls the opening and closing of the flow path of the liquid in the microchannel to the liquid flow control valve, and the microchannel is sequentially provided with a labeling zone and a detection zone from left to right, characterized in that, The marking area includes a first marking area arranged on the lower surface of the cover sheet and a second marking area arranged on the upper surface of the base sheet, the detection area includes an HEV-IgM antibody detection area arranged on the lower surface of the cover sheet and an HAV-IgM antibody detection area arranged on the upper surface of the base sheet, the HEV-IgM antibody detection area and the HAV-IgM antibody detection area are arranged along the microchannel from left to right, a sample injection hole is further arranged on the cover sheet, the sample injection hole is arranged between the detection area and the liquid flow control valve, an IgG antibody capture area is arranged on the upper surface of the base sheet corresponding to the sample injection hole, a magnetic control intercept valve is arranged between the marking area and the detection area of the microchannel, the magnetic control intercept valve controls the opening and closing of the flow path between the marking area and the detection area in the microchannel through magnet adsorption, the IgG antibody capture area is coated with magnetic microspheres-anti-human IgG antibody, the first marking area is coated with labeled HEV antigen, the second marking area is coated with labeled anti-human IgM antibody, the HEV-IgM antibody detection area is coated with anti-human IgM antibody, the HAV-IgM antibody detection area is coated with HAV antigen, a flow guide hole is arranged on the cover sheet, the bottom of the magnetic control intercept valve passes through the flow guide hole and contacts the upper surface of the base sheet, the top of the magnetic control intercept valve is made of ferrous material that can be adsorbed by a magnet, the bottom of the magnetic control intercept valve is separated from the flow guide hole after being adsorbed by the magnet, the bottom of the magnetic control intercept valve is made of water-absorbing material, and the water-absorbing force provided by the magnetic control intercept valve is greater than the capillary force in the microchannel.

2. The microfluidic chip for combined detection of IgM antibodies to hepatitis A and E viruses according to claim 1, characterized in that, The labeled anti-human IgM antibody is labeled mouse anti-human IgM antibody, the anti-human IgM antibody is mouse anti-human IgM antibody, and the HAV antigen is indirectly coated on the HAV-IgM antibody detection area through mouse anti-human IgM antibody.

3. The microfluidic chip for combined detection of IgM antibodies to hepatitis A and E viruses according to claim 1, characterized in that, The first marking area and the second marking area coincide in the vertical plane.

4. The microfluidic chip for combined detection of IgM antibodies to hepatitis A and E viruses according to claim 1, characterized in that, The horizontal distance between the HEV-IgM antibody detection area and the buffer injection hole is 30-35 mm, and the horizontal distance between the HAV-IgM antibody detection area and the buffer injection hole is 40-45 mm.

5. The microfluidic chip for combined detection of IgM antibodies to hepatitis A and E viruses according to claim 1, characterized in that, A groove is arranged on the lower surface of the cover sheet along the length direction, the cover sheet and the upper surface of the base sheet form a microchannel through the groove, and the width of the microchannel is 2-3 mm.

6. The microfluidic chip for combined detection of IgM antibodies to hepatitis A and E viruses according to claim 1, characterized in that, The labeled HEV antigen and the labeled anti-human IgM antibody are fluorescent microsphere labels.

7. The microfluidic chip for combined detection of IgM antibodies to hepatitis A and E viruses according to claim 1, characterized in that, The liquid flow control valve is movable water-absorbing material, and the water-absorbing material moves to contact or move away from the microchannel.

Images