Immune micro-fluidic chip for joint detection of IgMIgG antibodies of same pathogen and application of immune micro-fluidic chip

Abstract

The invention provides a method for preparing the same pathogen IgMamp; the immune micro-fluidic chip comprises a substrate and a cover plate pressed on the substrate, the substrate and the cover plate define a micro-channel, the micro-channel is sequentially provided with a marking area and a detection area from left to right, the detection area comprises an IgG detection area and an IgM detection area, the position, corresponding to the marking area, of the upper surface of the substrate is coated with a marked anti-human IgG antibody, and the IgM detection area is coated with an anti-human IgG antibody. The position, corresponding to the IgG detection area, of the upper surface of the substrate is coated with a pathogen recombinant antigen, the position, corresponding to the marking area, of the lower surface of the cover plate is coated with a marking antigen, and the position, corresponding to the IgM detection area, of the lower surface of the cover plate is coated with an anti-human IgM antibody. An IgM antibody capture detection area and an IgG antibody indirect detection area are respectively integrated on independent fluid layers of the chip, and synchronous but non-interfering detection of IgM and IgG antibodies in the same channel can be realized after a single sample is injected.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostics and immunoassay technology, and specifically relates to an immunomicrofluidic chip for the joint detection of IgM and IgG antibodies against the same pathogen and its application. Background Technology

[0002] After pathogens invade the body, they stimulate an immune response, which is divided into a primary immune response and a secondary immune response. The primary immune response has a long latency period, low antibody levels, and low affinity for the antigen. The earliest antibody to appear in this phase is IgM antibody, which can remain in the bloodstream for weeks or months. Later, IgG antibodies are produced, reaching their peak when IgM antibodies are nearing disappearance; these can remain in the bloodstream for years. In the terminal phase of the primary immune response, the antigen is cleared, a large number of plasma cells and effector T cells die, antibody levels gradually decline, and the body's immune system returns to its stable baseline state. When pathogens re-invade the body, memory lymphocytes can rapidly, specifically, and efficiently generate a secondary immune response. The secondary immune response has a short latency period, is mainly composed of IgG antibodies, has high concentrations, high affinity, and a long duration of action.

[0003] Simultaneous detection of pathogen-specific IgM and IgG antibodies is of significant value in clinical diagnosis. IgM antibodies are often a marker of early immune response, indicating recent or acute infection; while IgG antibodies typically indicate past or reinfection. Simultaneous detection of IgM and IgG antibodies helps physicians more accurately determine the stage of infection, providing crucial information for clinical treatment and prevention. Based on the IgM and IgG antibody test results, there are four possible combinations: ① IgM+ / IgG-: indicating early acute infection; ② IgM+ / IgG+: indicating mid-to-late acute infection or recent reinfection; ③ IgM- / IgG+: indicating past infection; ④ IgM- / IgG-: can help rule out infection or indicate the window period, requiring dynamic retesting. In summary, simultaneous detection of pathogen-specific IgM and IgG antibodies not only avoids the time window limitations of single indicators but also quantitatively assesses the infection progression through the antibody ratio (IgM / IgG).

[0004] There are various modes available for detecting pathogen-specific antibodies, among which the capture mode is a classic mode for detecting pathogen-specific IgM antibodies. Its detection principle is as follows: First, anti-human IgM antibodies are coated onto the surface of a solid-phase support. Second, the sample to be tested is added; all IgM antibodies in the sample are captured by the anti-human IgM antibodies on the solid-phase surface, resulting in an enrichment effect. After washing to remove unbound material, a labeled antigen reagent is added. This reagent binds only to the pathogen-specific IgM antibodies bound to the solid phase, forming a solid-phase anti-human IgM antibody-specific IgM antibody-labeled antigen complex. Finally, the free label is washed away, and the detection is performed.

[0005] The indirect mode is the classic mode for detecting pathogen-specific IgG antibodies: First, recombinant pathogen antigens are coated onto the surface of a solid-phase carrier. Second, the sample to be tested is added. If pathogen-specific IgG antibodies are present in the sample, they bind to the recombinant pathogen antigens on the solid-phase surface. After washing to remove unbound material, labeled anti-human IgG antibodies are added, forming a solid-phase antigen-specific IgG antibody-labeled anti-human IgG antibody complex.

[0006] When simultaneously detecting multiple targets in a conventional lateral flow microfluidic chip, the corresponding detection points are usually located on the same layer of the chip, leading to mutual interference when pathogen-specific IgM and IgG antibodies are detected simultaneously. For example, in a conventional unidirectional lateral flow microfluidic chip, when simultaneously detecting pathogen-specific IgM and IgG antibodies in the same microchannel, it is necessary to integrate capture and indirect modes. In this case, the labeled region contains labeled antigens and labeled anti-human IgG antibodies. Because the sample preferentially reacts with the labeled material, both pathogen-specific IgM and IgG antibodies in the sample can bind to the labeled antigens, and non-specific IgG antibodies can also bind to the labeled secondary antibodies. This results in low sensitivity due to various influencing factors.

[0007] CN221926380U discloses a microfluidic chip capable of bidirectional liquid flow. In this chip, when using a capture method and an indirect method to simultaneously detect pathogen-specific IgM and IgG antibodies, anti-human IgM antibodies and pathogen recombinant antigens are coated at the detection positions corresponding to IgM and IgG on the substrate, respectively. Labeled antigens and labeled anti-human IgG antibodies are placed in the detection area. After the sample to be tested is added to the right-side sample well, the sample flows to the left, sequentially passing through the IgM detection area and the IgG detection area. When flowing through the IgM detection area, the pathogen-specific IgM antibodies in the sample are captured by the solidified antibody. Subsequently, when the liquid flows through the IgG detection area, if the sample contains pathogen-specific IgG antibodies, they bind to the solid-phase antigen, and the pathogen IgM antibodies in the sample also bind to the solid-phase antigen at this location. Excess liquid flows through the guide hole into the waste liquid chamber. Figure 1A). After the pre-dried reagent in the labeled area is dissolved in buffer, it preferentially reaches the IgG detection area, allowing the labeled anti-human IgG antibody (labeled secondary antibody) to bind to the captured pathogen-specific IgG antibody. However, the labeled antigen subsequently used for IgM antibody detection will also bind to the captured pathogen-specific IgG antibody in this area, neutralizing some of the detection reagent and reducing the sensitivity of IgM detection. Furthermore, the labeled antigen may also bind to incorrectly bound IgM antibodies in this area, forming a double-antigen sandwich complex, leading to false positive signals in the IgG detection area. Figure 1 B). Summary of the Invention

[0008] This invention aims to provide an immunomicrofluidic chip for the joint detection of IgM and IgG antibodies against the same pathogen and its application. By optimizing the chip design and detection method, it solves the problems existing in the prior art and effectively avoids the mutual interference problem when pathogen-specific IgM / IgG antibodies are detected together in traditional detection.

[0009] To achieve the above objectives, the technical solution of the present invention is as follows: an immunomicrofluidic chip for the joint detection of IgM and IgG antibodies against the same pathogen, comprising a substrate and a cover plate pressed onto the substrate, wherein 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, which controls the opening and closing of the flow path of liquid in the microchannel to the flow control valve; the microchannel is provided with a labeling area and a detection area from left to right, the detection area including an IgG detection area and an IgM detection area; a sample injection hole is also provided on the cover plate, the sample injection hole being located between the detection area and the flow control valve; a magnetically controlled interceptor valve is provided within the microchannel. Between the labeling area and the detection area, the magnetically controlled interceptor 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 upper surface of the substrate is coated with labeled anti-human IgG antibody at the position corresponding to the labeling area, and the upper surface of the substrate is coated with pathogen recombinant antigen at the position corresponding to the IgG detection area. The lower surface of the cover plate is coated with labeled antigen at the position corresponding to the labeling area, and the lower surface of the cover plate is coated with anti-human IgM antibody at the position corresponding to the IgM detection area. The IgG detection area and the IgM detection area do not overlap in the vertical plane.

[0010] In one embodiment of the present invention, the IgG detection area and the IgM detection area are arranged sequentially from left to right along the microchannel.

[0011] In one embodiment of the present invention, the detection area includes multiple groups of IgG detection areas and IgM detection areas arranged from left to right along the microchannel, and each group of IgG detection areas and IgM detection areas corresponds to the detection of specific IgM / IgG antibodies against the same pathogen.

[0012] In one embodiment of the present invention, a groove is provided on the lower surface of the cover plate 2 along its length direction, and the cover plate 2 forms a microchannel 3 by means of the groove and the upper surface of the substrate 1, the width of the microchannel being 2-3 mm.

[0013] In one embodiment of the present invention, the horizontal distance from the IgG detection area to the buffer injection well is 30-35 mm, and the horizontal distance from the IgM detection area to the buffer injection well is 40-45 mm.

[0014] 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.

[0015] 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.

[0016] On the other hand, the present invention also provides an application of the microfluidic chip in any of the above technical solutions for the joint detection of IgM and IgG antibodies against the same pathogen, comprising at least the following steps: 1) Move the flow control valve to the far right so that it is not connected to the microchannel. After adding the sample to be tested into the sample injection hole, the sample 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. At this time, the upper liquid flow contacts the lower surface of the cover plate. When it reaches the IgM detection area, the IgM antibody contained in the sample is captured by the anti-human IgM antibody solidified here. The lower liquid flow contacts the upper surface of the substrate. The pathogen-specific IgG antibody contained in the sample binds to the pathogen recombinant antigen solidified in the detection area. After all the liquid flow in the channel has been collected into the magnetic intercept valve, remove the intercept valve. At this time, the microchannel is restored to the open state. 2) Move the flow control valve to the far left so that it is embedded in the microchannel. Add buffer solution to the buffer injection hole. The buffer solution enters the microchannel under the action of capillary driving force and flows forward in a laminar flow state. The upper layer of liquid flows into contact with the lower surface of the cover and dissolves the labeled antigen when it reaches the labeling area. When the labeled antigen reaches the IgM detection area with the liquid flow, it binds to the captured pathogen-specific IgM antibody to form an anti-human IgM antibody-pathogen-specific IgM antibody-labeled antigen complex. The lower layer of liquid flows into contact with the upper surface of the substrate and dissolves the labeled anti-human IgG antibody when it reaches the labeling area. The labeled anti-human IgG antibody then binds to the captured pathogen-specific IgG antibody in the detection area to form a pathogen recombinant antigen-pathogen-specific IgG antibody-labeled anti-human IgG antibody complex. The remaining liquid flow is collected into the flow control valve. 3) Read the signal values ​​of the IgG and IgM detection areas using conventional microfluidics.

[0017] The beneficial effects of the immunomicrofluidic chip for the combined detection of IgM and IgG antibodies against the same pathogen obtained through the above technical solution and its application are as follows: 1. Effectively improves the sensitivity of pathogen-specific IgM antibody detection: The IgM antibody capture detection area and the IgG antibody indirect detection area are integrated into separate fluid layers of the chip, and the microchannel height (20-50 μm) is precisely controlled to maintain a stable laminar flow. After a single sample is injected, IgM and IgG antibodies can be detected synchronously and without interference in the same channel. Since the labeled antigen moves closely to the lower surface of the cover with the upper fluid flow, it will not encounter the IgG antibody on the upper surface of the substrate, thus effectively avoiding the neutralization effect of IgG antibody on the labeled antigen.

[0018] 2. Effectively improves the specificity of pathogen-specific IgG antibody detection: When the lower liquid stream contacts the upper surface of the substrate and reaches the labeled area, it dissolves the labeled anti-human IgG antibody there. Subsequently, it binds to the pathogen-specific IgG antibody already captured in the detection area. At this point, even if some IgM antibody is mistakenly bound to the IgG detection area, it will not bind to the subsequent labeled anti-human IgG antibody. In addition, because the labeled antigen moves closely to the top layer of the chip with the upper liquid stream, it will not encounter the IgM antibody on the bottom layer of the chip, thus effectively avoiding the problem of false positive signals in the IgG detection area. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the traditional bidirectional microfluidic chip detection principle in the background technology of this invention; Figure 2 This is a schematic diagram of the structure of the immunomicrofluidic chip (transparent) for the joint detection of IgM and IgG antibodies against the same pathogen as described in this invention; Figure 3This is a schematic diagram of the cover plate (transparent) of the present invention; Figure 4 This is a schematic diagram of the structure of the substrate described in this invention; Figure 5 This is a cross-sectional view of the immunomicrofluidic chip for the combined detection of IgM and IgG antibodies against the same pathogen as described in this invention. Figure 6 This is a schematic diagram of the liquid flow state in the microchannel described in this invention; Figure 7 This is a schematic diagram of the principle of adding a sample to be tested using a microfluidic chip based on laminar flow as described in this invention; Figure 8 This is a schematic diagram of the principle of adding buffer solution using a microfluidic chip based on laminar flow as described in this invention; Figure 9 This is a schematic diagram illustrating the principle of adding a sample to be tested when the microfluidic chip described in this invention performs combined detection of IgM and IgG antibodies against the same pathogen. Figure 10 This invention is in Figure 9 A schematic diagram of the reaction stage of the sample under test on the chip; Figure 11 This invention is in Figure 10 A schematic diagram showing the principle of the sample to be tested on the chip absorbing waste liquid through a magnetically controlled interception valve; Figure 12 This invention is in Figure 11 A schematic diagram of the principle of adding buffer solution onto the chip; Figure 13 This is a schematic diagram of the structure of the top laminar flow verification of biomolecule distribution on the microfluidic chip based on laminar flow described in this invention; Figure 14 This is a schematic diagram of the structure of the microfluidic chip based on laminar flow for verifying the distribution of biomolecules at the bottom layer, as described in this invention. Figure 15 This is a schematic diagram of the structure of a microfluidic chip in one embodiment of the present invention for the combined detection of IgM and IgG antibodies against Chikungunya and dengue viruses.

[0020] In the figure, 1 is the substrate; 2 is the cover plate; 3 is the microchannel; 4 is the buffer injection port; 5 is the flow control valve; 6 is the sample injection port; 7 is the magnetically controlled intercept valve; 31 is the labeling area; 32 is the IgG detection area; and 33 is the IgM detection area. Detailed Implementation

[0021] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0022] 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.

[0023] This invention relates to the field of immunoassay technology, specifically to an immunomicrofluidic chip for the joint detection of IgM and IgG antibodies against the same pathogen and its application. Addressing the bottleneck issue of mutual interference between IgM and IgG antibodies in existing pathogen antibody co-detection technologies, this invention proposes a layered microfluidic chip. It employs a capture method to detect specific IgM antibodies and an indirect method to detect specific IgG antibodies. By integrating the IgM antibody capture detection area and the IgG antibody indirect detection area into separate fluid layers of the chip, simultaneous but non-interfering detection of IgM and IgG antibodies in the same channel can be achieved after a single sample injection.

[0024] 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.

[0025] like Figure 2-5 As shown, this invention proposes an immunomicrofluidic chip for the joint detection of IgM and IgG antibodies against the same pathogen, 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 μm. The left end of the microchannel 3 is connected to a buffer injection port 4 on the cover plate 2, and a flow control valve 5 is provided at the right end of the microchannel 3. 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. From left to right, the microchannel 3 is provided with a labeling area 31 and a detection area, the detection area including an IgG detection area 32 and an IgM detection area 33. The cover plate 2 is also provided with a sample injection port 6, which is located between the detection area and the flow control valve 5. A magnetically controlled intercepting valve 7 is also provided in the microchannel 3, located between the labeling area 31 and the detection area. Between the regions, the magnetically controlled intercept valve 7 uses a magnet to attract and control the opening and closing of the flow path between the labeling region 31 and the detection region in the microchannel 3. The upper surface of the substrate 1 (bottom layer of the chip) is coated with labeled anti-human IgG antibody at the position corresponding to the labeling region 31. The upper surface of the substrate 1 (bottom layer of the chip) is coated with pathogen recombinant antigen at the position corresponding to the IgG detection region 32. The lower surface of the cover plate 2 (top layer of the chip) is coated with labeled antigen at the position corresponding to the labeling region 31. The lower surface of the cover plate 2 (top layer of the chip) is coated with anti-human IgM antibody at the position corresponding to the IgM detection region 32. The IgG detection region 31 and the IgM detection region 32 do not overlap on the vertical plane.

[0026] The method employs a capture method to detect specific IgM antibodies and an indirect method to detect specific IgG antibodies. By integrating the IgM antibody capture detection area and the IgG antibody indirect detection area into separate fluid layers of the chip and precisely controlling the height of the microchannels to maintain a stable laminar flow, simultaneous but non-interfering detection of IgM and IgG antibodies can be achieved in the same channel after a single sample injection. Specifically, pathogen-specific IgM antibodies in the upper layer of the fluid flow are specifically captured by the anti-human IgM antibody pre-coated on the top layer of the chip and bind to the labeled antigen, while pathogen-specific IgG antibodies in the lower layer of the fluid flow are specifically captured by the recombinant antigen pre-coated on the bottom layer of the chip and bind to the labeled anti-human IgG antibody. Spatial separation between the IgM and IgG antibody detection areas eliminates the risk of binding to non-target antibodies, effectively avoiding the mutual interference problem in the joint detection of pathogen-specific IgM / IgG antibodies in traditional detection.

[0027] 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.

[0028] 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.

[0029] like Figure 6 As shown, 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 can ensure that any liquid flow exhibits a stable laminar flow state within the microchannel.

[0030] The working procedure of the layered microfluidic chip is as follows: Detection molecule capture stage: such as Figure 7As shown, the flow control valve 5 is switched to the far right, disconnecting it from the microchannel 3. After the sample to be tested is added into the sample injection port, the sample flows to the left and right. Since the microchannel 3 is not connected to the flow control valve 5, the liquid flowing to the right remains at the end of the microchannel 3 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 antigen / antibody in the detection area. When the liquid continues to flow and reaches the magnetically controlled intercept valve 7, it is intercepted. Because the water absorption force provided by the magnetically controlled intercept valve 7 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. After all the liquid in the microchannel has been collected into the magnetically controlled intercept valve 7, the intercept valve is removed, and the microchannel is restored to unobstructed flow, ensuring that the subsequent reagent flow and reaction are not disturbed.

[0031] B. The stage of marker substance binding: such as Figure 8 As shown, move the flow control valve to the far left so that it is embedded in the microchannel. Add buffer solution to the buffer injection hole. Under the action of capillary driving force, the buffer solution enters the microchannel and dissolves the labeled substance in the labeled area as it flows through it. After the labeled substance reaches the detection area with the liquid flow, it is captured by the corresponding substance in the detection area, and the remaining liquid flow is collected into the flow control valve.

[0032] The IgG detection area and the IgM detection area are arranged sequentially from left to right along the microchannel.

[0033] The detection area includes multiple groups of IgG detection areas 32 and IgM detection areas 33 arranged from left to right along the microchannel. Each group of IgG detection areas 32 and IgM detection areas 33 corresponds to the detection of specific IgM / IgG antibodies of the same pathogen, which can be applied to the joint detection of IgM / IgG specific antibodies of multiple pathogens.

[0034] 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 is 2-3 mm.

[0035] The horizontal distance from the IgG detection area to the buffer injection well is 30-35 mm, and the horizontal distance from the IgM detection area to the buffer injection well is 40-45 mm.

[0036] 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.

[0037] The flow control valve 5 is made of a movable absorbent material, which moves to contact or move away from the microchannel 3.

[0038] The principle of combined detection of pathogen-specific IgM and IgG is as follows. Detection molecule capture stage: like Figure 9 As shown, move the flow control valve to the far right to disconnect it from the microchannel. Figure 10 As shown, after the sample is added to the sample injection hole, the sample flows to the left and right. Since the microchannel is not connected to the flow control valve at this time, 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. The upper liquid flow contacts the lower surface of the cover plate (top layer of the chip). When it reaches the IgM detection area, the IgM antibody contained in the sample is captured by the anti-human IgM antibody solidified here, and the remaining substances are collected into the magnetically controlled intercept valve. The lower liquid flow contacts the upper surface of the substrate (bottom layer of the chip). The pathogen-specific IgG antibody contained in the sample binds to the pathogen recombinant antigen solidified in the detection area. However, the pathogen-specific IgM antibody contained in the sample can also bind to the pathogen recombinant antigen here. Due to the difference in affinity between IgG antibody and IgM antibody, IgG antibody will preferentially bind to the recombinant antigen. Figure 11 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.

[0039] Labeling process: like Figure 12 As shown, the flow control valve is switched to the far left, embedding it within the microchannel. Buffer solution is added to the buffer injection well, entering the microchannel under capillary force and flowing forward in a laminar flow. The upper layer of the flow contacts the inner surface of the chip's top layer, dissolving the labeled antigen in the labeled area. When the labeled antigen reaches the IgM detection area, it binds to the captured pathogen-specific IgM antibodies, forming an anti-human IgM antibody-pathogen-specific IgM antibody-labeled antigen complex. The lower layer of the flow contacts the inner surface of the chip's bottom layer, dissolving the labeled anti-human IgG antibodies in the labeled area, and subsequently binding to the captured pathogen-specific IgG antibodies in the detection area. At this point, even if some pathogen-specific IgM antibodies mistakenly bind to the IgG detection area, they will not bind to subsequent labeled anti-human IgG antibodies. Furthermore, because the labeled antigen moves closely to the chip's top layer with the upper layer of the flow, it will not encounter IgM antibodies on the chip's bottom layer, effectively avoiding false positive signals caused by IgM in the IgG detection area.

[0040] It is understood that the present invention does not limit the placement of the IgG detection area 32 on the upper surface of the substrate 1 (bottom layer of the chip) and the IgM detection area 32 on the lower surface of the cover plate (top layer of the chip). If the position of the labeling area 31 on the lower surface of the cover plate 2 (top layer of the chip) is coated with labeled anti-human IgG antibody, the position of the IgG detection area 32 on the lower surface of the cover plate 2 (top layer of the chip) is coated with pathogen recombinant antigen, the position of the labeling area 31 on the upper surface of the substrate 1 (bottom layer of the chip) is coated with labeled antigen, and the position of the IgM detection area 32 on the upper surface of the substrate 1 (bottom layer of the chip) is coated with anti-human IgM antibody, it is also possible to integrate the IgM antibody capture detection area and the IgG antibody indirect detection area on separate fluid layers of the chip.

[0041] Example 1: In-Channel Laminar Flow Verification of a Chip 1. Chip fabrication Both the chip substrate and cover plate are made of PMMA material. The microchannel structure, sample injection port, buffer injection port, drainage port and other structures of the cover plate are designed using CAD software, and then the PMMA surface is processed using a CO2 laser etching machine.

[0042] 2. Biomolecular coating procedures First, 1.5 μL of streptavidin was spotted at the IgM detection area on the top or bottom layer of the chip, incubated for 1 h, washed, and dried. Biotinylated anti-human IgM antibody was then spotted at the IgM detection area, and incubation continued for 1 h. After another wash, fluorescent microspheres conjugated with MP recombinant antigen were spotted at the labeled area on the top or bottom layer of the chip and dried at 37°C.

[0043] 3. Microfluidic chip assembly procedure First, the magnetically controlled interceptor valve is embedded in the liquid guiding hole on the top layer of the chip; second, the liquid flow control valve is placed in the waste liquid chamber on the top layer of the chip; finally, according to the verification purpose, the top and bottom layers of the chip are assembled and bonded (i.e., the labeled area and the detection area are located on the same level in the experimental group, and the labeled area and the detection area are located on different levels in the control group).

[0044] The distribution of biomolecules in the top layer of the chip, as verified by laminar flow, is shown in Table 1-1. The specific structure is as follows: Figure 13 As shown in the figure, A is the experimental group and B is the control group.

[0045] Table 1-1 Chip Top-Level Laminar Flow Verification

[0046] The distribution of biomolecules at the bottom layer of the chip, as verified by laminar flow, is shown in Table 1-2. The specific structure is as follows: Figure 14 As shown in the figure, A is the experimental group and B is the control group.

[0047] Table 1-2 Chip Bottom Layer Flow Verification

[0048] 4. Testing Procedure First, move the flow control valve to the far right, disconnecting it from the microchannel. Add 10 μL of the sample to be tested into the sample injection port. The sample flows to the left and right. Because 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 tip and will not flow out spontaneously, while the liquid flowing to the left continues to flow. When it reaches the detection zone, the MP-specific IgM antibody in the sample is captured by the anti-human IgM antibody in the detection zone. The liquid continues to flow and is intercepted at the magnetically controlled intercept valve. Because the suction 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. Next, after all the liquid in the microchannel has been collected into the magnetically controlled intercept valve, remove the intercept valve. The microchannel is now unobstructed, ensuring that subsequent reagent flow and reactions are not disturbed. Next, move the flow control valve to its leftmost position, embedding it within the microchannel. Add buffer solution through the buffer injection well; driven by capillary force, the solution enters the microchannel and, as it flows through the labeled area, dissolves the fluorescent microspheres coupled with the MP recombinant antigen. The labeled substance, carried by the flow, reaches the detection area and binds to the specific IgM antibody already captured there. The remaining flow is collected in the flow control valve. Finally, use the analyzer to routinely read the fluorescence signal value of the detection area.

[0049] 5. Experimental Results Table 2-1 Chip Top-Level Verification Results

[0050] Table 2-2 Chip-level verification results

[0051] Experimental data shows that the detection zone can only detect the analyte signal when the labeling zone and the detection zone are set on the same level. When the labeling zone and the detection zone are set on different levels, the substance in the detection zone cannot capture the labeling substance, resulting in the detection zone not detecting the analyte signal. This confirms that the liquid flow in the microchannel is in a laminar flow state.

[0052] Example 2: Combined detection of MP-IgM and IgG antibodies 1. Chip fabrication Both the top and bottom layers of the chip were fabricated using PMMA material. The microchannel structure, sample injection port, buffer injection port, flow channel, and other structures of the top layer were designed using CAD software, and then the PMMA surface was processed using a CO2 laser etching machine.

[0053] 2. Biomolecular coating program on the top layer of the chip First, 1.5 μL of streptavidin was spotted on the top layer of the chip corresponding to the IgM antibody detection area. After incubation for 1 h and washing, the chip was dried and then biotinylated anti-human IgM antibody was spotted on the IgM antibody detection area. Incubation continued for another 1 h. After washing again, fluorescent microspheres conjugated with MP recombinant antigen were spotted on the labeled area and dried at 37°C.

[0054] 3. Biomolecular coating program at the chip's underlying layer First, 1.5 μL of streptavidin was spotted at the location corresponding to the IgG antibody detection area on the bottom layer of the chip, incubated for 1 h, washed, and dried. Biotinylated MP recombinant antigen was then spotted at the IgG antibody detection area, and incubation continued for 1 h. After another wash, fluorescent microspheres conjugated with anti-human IgG antibody were spotted at the labeled area and dried at 37°C.

[0055] Note: For traditional bidirectional microfluidic chips, both the IgM antibody detection area and the IgG antibody detection area are located at the bottom layer of the chip, and the biomolecule coating procedure is the same as above.

[0056] 4. Microfluidic chip assembly procedure 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.

[0057] 5. Testing Procedure Two samples, S1 and S2, were selected and tested using a conventional bidirectional flow microfluidic chip and the dual-layer microfluidic chip of this application, respectively. The conventional bidirectional flow microfluidic chip test is shown in CN221926380U, and the test steps of the dual-layer microfluidic chip of this application are the same as in Example 1.

[0058] 6. Experimental Results Table 3 Detection results of MP-IgM and IgG

[0059] For sample S1, the clinical laboratory detected MP-IgM antibody positive and MP-IgG antibody negative using magnetic microparticle chemiluminescence method. The detection results of the novel bilayer microfluidic chip proposed in this invention are consistent with the clinical test results. However, the detection results of conventional bidirectional microfluidic chips are positive for both MP-IgM antibody and MP-IgG antibody. This is because in conventional bidirectional microfluidic chips, the detection areas of IgM antibody and IgG antibody are located on the same layer. Some MP-IgM antibody binds to the IgG detection area and then binds to the subsequent labeled antigen, resulting in a false positive signal in the IgG detection area. This will cause clinicians to misjudge the stage of infection in the patient.

[0060] For sample S2, the clinical laboratory detected a weak positive result for MP-IgM antibody and a positive result for MP-IgG antibody using the magnetic microparticle chemiluminescence method. The detection results of the novel bilayer microfluidic chip proposed in this invention are consistent with the clinical test results. However, the detection results of the conventional bidirectional microfluidic chip are negative for MP-IgM antibody and positive for MP-IgG antibody. This is because in the conventional bidirectional microfluidic chip, the detection areas of IgM antibody and IgG antibody are located on the same layer, and the labeled substance preferentially reaches the IgG antibody detection area, causing some of the labeled antigen to be neutralized by the IgG antibody, thus resulting in a false negative result for low-concentration IgM antibody detection.

[0061] Example 3: Combined detection of IgM and IgG antibodies against Chikungunya and Dengue viruses 1. Chip Structure like Figure 15 As shown, the top layer of the chip is used for the detection of chikungunya virus IgM antibody (T2 detection area) and dengue virus IgG antibody (T4 detection area); the bottom layer of the chip is used for the detection of chikungunya virus IgG antibody (T1 detection area) and dengue virus IgM antibody (T3 detection area).

[0062] 2. Chip fabrication Both the top and bottom layers of the chip were fabricated using PMMA material. The microchannel structure, sample injection port, buffer injection port, flow channel, and other structures of the top layer were designed using CAD software, and then the PMMA surface was processed using a CO2 laser etching machine.

[0063] 3. Biomolecular coating program on the top layer of the chip First, 1.5 μL of streptavidin was sequentially spotted into the T2 and T4 detection zones on the top layer of the chip. After incubation for 1 h and washing, the chips were dried. Biotinylated anti-human IgM antibody was spotted into the T2 detection zone, and biotinylated dengue recombinant antigen was spotted into the T4 detection zone. Incubation was continued for another 1 h. After washing again, fluorescent microspheres conjugated with chikungunya recombinant antigen and fluorescent microspheres conjugated with anti-human IgG antibody were spotted into the labeled areas and dried at 37°C.

[0064] 4. Biomolecular coating program at the chip's underlying layer 1.5 μL of streptavidin was sequentially spotted in the T1 and T3 detection zones on the bottom layer of the chip, incubated for 1 h, washed, and dried. Biotinylated chikungunya recombinant antigen was spotted in the T1 detection zone, and biotinylated anti-human IgM antibody was spotted in the T4 detection zone. Incubation continued for 1 h. After another wash, fluorescent microspheres conjugated with dengue recombinant antigen and fluorescent microspheres conjugated with anti-human IgG antibody were spotted in the labeled areas and dried at 37°C.

[0065] 5. Microfluidic chip assembly procedure 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.

[0066] 6. Testing Procedure Three different samples, S1, S2, and S3, were selected for testing, and the testing steps were the same as in Example 1.

[0067] 7. Experimental Results Table 4 Clinical Sample Detection Results

[0068] 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.

[0069] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0070] 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.

[0071] 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. An immunomicrofluidic chip for the combined detection of IgM and IgG antibodies against the same pathogen, comprising a substrate and a cover sheet pressed onto the substrate, wherein the substrate and the cover sheet enclose a microchannel, characterized in that, The microchannel has a height of 20-50 μm. The left end of the microchannel communicates with a buffer injection port on the cover plate. A flow control valve is located at the right end of the microchannel, controlling the flow path of liquid within the microchannel. From left to right, the microchannel contains a labeling area and a detection area, including IgG and IgM detection areas. A sample injection port is also located on the cover plate between the detection area and the flow control valve. A magnetically controlled interceptor valve is installed within the microchannel. Between the labeling area and the detection area, the magnetically controlled interceptor 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 upper surface of the substrate is coated with labeled anti-human IgG antibody at the position corresponding to the labeling area, and the upper surface of the substrate is coated with pathogen recombinant antigen at the position corresponding to the IgG detection area. The lower surface of the cover plate is coated with labeled antigen at the position corresponding to the labeling area, and the lower surface of the cover plate is coated with anti-human IgM antibody at the position corresponding to the IgM detection area. The IgG detection area and the IgM detection area do not overlap in the vertical plane.

2. The immunomicrofluidic chip for combined detection of IgM and IgG antibodies against the same pathogen according to claim 1, characterized in that, The IgG detection area and the IgM detection area are arranged sequentially from left to right along the microchannel.

3. The immunomicrofluidic chip for combined detection of IgM and IgG antibodies against the same pathogen according to claim 1, characterized in that, The detection area includes multiple groups of IgG detection areas and IgM detection areas arranged from left to right along the microchannel, and each group of IgG detection areas and IgM detection areas corresponds to the detection of specific IgM and IgG antibodies against the same pathogen.

4. The immunomicrofluidic chip for combined detection of IgM and IgG antibodies against the same pathogen according to claim 1, characterized in that, The cover plate has a groove along its length on its lower surface. The cover plate forms a microchannel by enclosing the upper surface of the substrate through the groove. The width of the microchannel is 2-3 mm.

5. The immunomicrofluidic chip for combined detection of IgM and IgG antibodies against the same pathogen according to claim 2, characterized in that, The horizontal distance from the IgG detection area to the buffer injection well is 30-35 mm, and the horizontal distance from the IgM detection area to the buffer injection well is 40-45 mm.

6. The immunomicrofluidic chip for combined detection of IgM and IgG antibodies against the same pathogen according to claim 1, characterized in that, 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. After the magnetically controlled intercept valve is attracted by magnetic force, the bottom of the magnetically controlled intercept valve is separated from the flow guide hole. The bottom of the magnetically controlled intercept valve is provided with a water-absorbing material.

7. The immunomicrofluidic chip for combined detection of IgM and IgG antibodies against the same pathogen according to claim 1, characterized in that, The flow control valve is made of a movable absorbent material, which moves to contact or move away from the microchannel.

8. The application of an immunomicrofluidic chip for the joint detection of IgM and IgG antibodies against the same pathogen, characterized in that, The detection using the microfluidic chip according to any one of claims 1-7 includes at least the following steps: 1) Move the flow control valve to the far right so that it is not connected to the microchannel. After adding the sample to be tested into the sample injection hole, the sample 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. At this time, the upper liquid flow contacts the lower surface of the cover plate. When it reaches the IgM detection area, the IgM antibody contained in the sample is captured by the anti-human IgM antibody solidified here. The lower liquid flow contacts the upper surface of the substrate. The pathogen-specific IgG antibody contained in the sample binds to the pathogen recombinant antigen solidified in the detection area. After all the liquid flow in the channel has been collected into the magnetic intercept valve, remove the intercept valve. At this time, the microchannel is restored to the open state. 2) Move the flow control valve to the far left so that it is embedded in the microchannel. Add buffer solution to the buffer injection hole. The buffer solution enters the microchannel under the action of capillary driving force and flows forward in a laminar flow state. The upper layer of liquid flows into contact with the lower surface of the cover and dissolves the labeled antigen when it reaches the labeling area. When the labeled antigen reaches the IgM detection area with the liquid flow, it binds to the captured pathogen-specific IgM antibody to form an anti-human IgM antibody-pathogen-specific IgM antibody-labeled antigen complex. The lower layer of liquid flows into contact with the upper surface of the substrate and dissolves the labeled anti-human IgG antibody when it reaches the labeling area. The labeled anti-human IgG antibody then binds to the captured pathogen-specific IgG antibody in the detection area to form a pathogen recombinant antigen-pathogen-specific IgG antibody-labeled anti-human IgG antibody complex. The remaining liquid flow is collected into the flow control valve. 3) Read the signal values ​​of the IgG and IgM detection areas using conventional microfluidics.

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