Immunomicrofluidic chip for combined detection of igm & igg antibodies of same pathogen and application thereof

Abstract

This invention proposes an immunomicrofluidic chip for the joint detection of IgM and IgG antibodies against the same pathogen and its application. The chip includes a substrate and a cover plate pressed onto the substrate. The substrate and cover plate together form a microchannel. From left to right, the microchannel has a labeled area and a detection area. The detection area includes an IgG detection area and an IgM detection area. The upper surface of the substrate, corresponding to the labeled area, is coated with labeled anti-human IgG antibody, and the upper surface of the substrate, corresponding to the IgG detection area, is coated with recombinant pathogen antigen. The lower surface of the cover plate, corresponding to the labeled area, is coated with labeled antigen, and the lower surface of the cover plate, corresponding to the IgM detection area, is coated with anti-human IgM antibody. 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.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of in vitro diagnosis and immunodetection, and particularly relates to an immunomicrofluidic chip for combined detection of IgM and IgG antibodies of the same pathogen and application thereof. BACKGROUND

[0002] After the invasion of pathogens into the body, the body starts an immune response, which is divided into primary immune response and secondary immune response. The primary immune response produces antibodies with a long incubation period, a small amount of antibodies, and a low affinity for antigen binding. The earliest antibody appearing in this period is IgM antibody, which can be maintained in the blood for several weeks or months. IgG antibodies can be produced in the later period, and when IgM antibodies approach extinction, IgG antibodies reach a peak period, which can be maintained in the blood for a long time, up to several years. At the end of the primary immune response, the antigen is cleared, a large number of plasma cells and effector T cells die, and the antibody level in the body gradually decreases, and the immune system of the body returns to its stable basic state. When the pathogen invades the body again, the memory lymphocytes in the body can quickly, specifically and efficiently produce a secondary immune response. The secondary immune response produces antibodies with a short incubation period, mainly IgG antibodies, high antibody concentration, high affinity, and long duration.

[0003] The simultaneous detection of pathogen-specific IgM and IgG antibodies has important value in clinical diagnosis. IgM antibodies are usually a marker of early immune response and are often used to indicate recent or acute infection; while IgG antibodies usually indicate past infection or reinfection. By simultaneously detecting IgM and IgG antibodies, doctors can more accurately determine the infection stage of patients, thereby providing an important basis for clinical treatment and prevention. According to the detection results of IgM and IgG antibodies, there are the following four combination modes: ① IgM+ / IgG-: indicating the early stage of acute infection; ② IgM+ / IgG+: indicating the middle and late stages of acute infection or recent reinfection; ③ IgM- / IgG+: indicating past infection; ④ IgM- / IgG-: which can help exclude infection or indicate the window period and requires dynamic re-examination. In summary, the simultaneous detection of pathogen-specific IgM and IgG antibodies not only avoids the time window limitations of a single indicator, but also quantitatively evaluates the infection process through the antibody ratio (IgM / IgG).

[0004] There are various modes that can be used for pathogen-specific antibody detection, among which the capture mode is a classic mode for pathogen-specific IgM antibody detection, and the detection principle is as follows: first, anti-human IgM antibodies are coated on the surface of a solid carrier. Second, the sample to be tested is added, and the IgM antibodies in the sample to be tested are captured by the anti-human IgM antibodies on the solid surface, and an enrichment effect is achieved. After washing to remove the unbound substances, a labeled antigen reagent is added, which only binds to the pathogen-specific IgM antibodies bound to the solid phase to form a solid phase anti-human IgM antibody-specific IgM antibody-labeled antigen complex. Finally, the free label is removed by washing and detected.

[0005] The indirect mode is a classic mode for pathogen-specific IgG antibody detection: first, pathogen recombinant antigens are coated on the surface of a solid carrier. Second, the sample to be tested is added, and if pathogen-specific IgG antibodies are present in the sample to be tested, the pathogen-specific IgG antibodies will bind to the pathogen recombinant antigens on the solid surface. After washing to remove the unbound substances, a labeled anti-human IgG antibody is added to form a solid phase antigen-specific IgG antibody-labeled anti-human IgG antibody complex.

[0006] When multiple targets are detected simultaneously in a conventional lateral flow microfluidic chip, the corresponding detection points are usually arranged on the same plane of the chip, which causes mutual influence when pathogen-specific IgM and IgG antibodies are detected simultaneously. For example, in a conventional one-way lateral flow microfluidic chip, when pathogen-specific IgM and IgG antibodies are detected simultaneously in the same microchannel, the capture mode and the indirect mode need to be integrated, and the labeling region contains labeled antigens and labeled anti-human IgG antibodies. Because the sample reacts with the substances in the labeling region first, the pathogen-specific IgM and IgG antibodies in the sample can both bind to the labeled antigens, and non-specific IgG antibodies can also bind to the labeled secondary antibody, which is affected by multiple factors and has low sensitivity.

[0007] CN221926380U discloses a microfluidic chip that can realize bidirectional flow of liquid. In this chip, when pathogen-specific IgM and IgG antibodies are detected simultaneously using the capture method and the indirect method, anti-human IgM antibodies and pathogen recombinant antigens are coated on the detection positions corresponding to IgM and IgG in the detection region of the substrate, respectively, and labeled antigens and labeled anti-human IgG antibodies are placed in the detection region. After the sample to be tested is added to the right sample addition hole, the sample flows leftward through the IgM detection region and the IgG detection region in sequence. When flowing through the IgM detection region, the pathogen-specific IgM antibodies in the sample are captured by the solidified antibodies; then, when the liquid flow flows through the IgG detection region, if the sample contains pathogen-specific IgG antibodies, they will bind to the solid phase antigens, and the pathogen IgM antibodies in the sample will also bind to the solid phase antigens here, and the excess liquid flows into the waste liquid chamber through the flow guide hole. Figure 1A). After adding the pre-dried reagent of the labeling area to dissolve the buffer solution, it preferentially reaches the IgG detection area, so that the labeled anti-human IgG antibody (labeled secondary antibody) is combined with the captured pathogen-specific IgG antibody, and the labeled antigen used for subsequent IgM antibody detection is also combined with the captured pathogen-specific IgG antibody, neutralizing part of the detection reagent and reducing the sensitivity of IgM detection; in addition, the labeled antigen is also combined with the IgM antibody to form a double-antigen sandwich complex, resulting in a false positive signal in the IgG detection area Figure 1 B). SUMMARY

[0008] The present application aims to provide an immunomicrofluidic chip for combined detection of IgM and IgG antibodies of the same pathogen and its application. By optimizing the design of the chip and the detection method, the problems existing in the prior art are solved, and the mutual influence problem in the combined detection of pathogen-specific IgM / IgG antibodies in traditional detection is effectively avoided.

[0009] To achieve the above-mentioned purpose, the technical scheme of the present application is as follows: an immunomicrofluidic chip for combined detection of IgM and IgG antibodies of the same pathogen, 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 opened on the cover plate, a liquid flow control valve is arranged at the right end of the microchannel, 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, the microchannel is sequentially provided with a labeling area and a detection area from left to right, the detection area comprises an IgG detection area and an IgM detection area, a sample injection hole is further arranged on the cover plate, the sample injection hole is arranged between the detection area and the liquid flow control valve, a magnetic control interception valve is arranged in the microchannel, the magnetic control interception valve is arranged between the labeling area and the detection area, the magnetic control interception valve controls the opening and closing of the flow path between the labeling area and the detection area in the microchannel through a magnet, the upper surface of the substrate is coated with labeled anti-human IgG antibody at the position corresponding to the labeling area, 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, the lower surface of the cover plate is coated with anti-human IgM antibody at the position corresponding to the IgM detection area, and the IgG detection area and the IgM detection area do not coincide in the vertical plane.

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

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

[0012] In one embodiment of the present application, the lower surface of the cover sheet 2 is provided with a groove along the length direction thereof, and the microchannel 3 is formed by the groove and the upper surface of the substrate 1.

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

[0014] In one embodiment of the present application, the cover sheet is provided with a flow guide hole, the bottom of the magnetic control interception valve passes through the flow guide hole and contacts the upper surface of the substrate, the top of the magnetic control interception valve is provided with ferrous material that can be attracted by a magnet, the magnetic control interception valve is attracted by magnetic force and the bottom thereof is separated from the flow guide hole, and the bottom of the magnetic control interception valve is provided with water-absorbing material.

[0015] In one embodiment of the present application, the liquid flow control valve is movable water-absorbing material, and the water-absorbing material moves to contact or move away from the microchannel.

[0016] In another aspect, the present application also provides a use of the microfluidic chip in any one of the above technical solutions for combined detection of IgM & IgG antibodies of the same pathogen, comprising at least the following steps:

[0017] 1) Move the liquid flow control valve to the rightmost position so that it is not connected to the microchannel, after adding the sample to be tested to the sample injection hole, the sample flows to the left and right sides, the liquid flowing to the right stays at the end of the microchannel under the action of surface tension, and the liquid flowing to the left can continue to flow, at this time, the upper layer of liquid flow contacts the lower surface of the cover sheet, when reaching the IgM detection zone, the IgM antibodies contained in the sample are captured by the anti-human IgM antibodies solidified there, the lower layer of liquid flow contacts the upper surface of the substrate, and the pathogen-specific IgG antibodies contained in the sample bind to the pathogen recombinant antigens solidified in the detection zone, after all the liquid flow in the channel is collected into the magnetic control interception valve, the interception valve is removed, at this time, the microchannel returns to the state of being unobstructed.

[0018] 2) Turn the liquid flow control valve to the leftmost position, so that it is embedded in the microchannel, and add buffer solution at the buffer injection hole, which enters the microchannel under the action of capillary force, and flows forward in a laminar flow state. The upper layer of liquid flow contacts the lower surface of the cover sheet, and the labeled antigen is dissolved at the marker area. When the labeled antigen reaches the IgM detection area, 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 flow contacts the upper surface of the substrate, and the labeled anti-human IgG antibody is dissolved at the marker area, and 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, and the remaining liquid flow is collected in the liquid flow control valve;

[0019] 3) Read the signal values of the IgG detection area and the IgM detection area in the conventional microfluidic chip.

[0020] The immunomicrofluidic chip for combined detection of IgM & IgG antibodies of the same pathogen and its application obtained by the above technical solution have the beneficial effects of:

[0021] 1. The sensitivity of pathogen-specific IgM antibody detection can be effectively improved: the IgM antibody capture detection area and the IgG antibody indirect detection area are integrated in separate fluid layers of the chip, and the microchannel height (20-50 μm) is precisely controlled to maintain a stable laminar flow state of the liquid flow. After a single sample is injected, the IgM and IgG antibodies can be detected simultaneously and without interference in the same channel. Since the labeled antigen moves closely to the lower surface of the cover sheet with the upper layer of liquid flow, it does not meet the IgG antibody on the upper surface of the substrate, thereby effectively avoiding the neutralization effect of the IgG antibody on the labeled antigen.

[0022] 2. The specificity of pathogen-specific IgG antibody detection can be effectively improved: the lower layer of liquid flow contacts the upper surface of the substrate, and the labeled anti-human IgG antibody is dissolved at the marker area, and then binds to the captured pathogen-specific IgG antibody in the detection area. At this time, even if part of the IgM antibody is incorrectly combined in the IgG detection area, it will not bind to the subsequent labeled anti-human IgG antibody. In addition, since the labeled antigen moves closely to the top layer of the chip with the upper layer of liquid flow, it does not meet the IgM antibody on the bottom layer of the chip, thereby effectively avoiding the problem of false positive signals in the IgG detection area. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is the traditional bidirectional microfluidic chip detection principle diagram in the background art of the present application;

[0024] Figure 2The structure diagram of the immunomicrofluidic chip (perspective) for the combined detection of IgM & IgG antibodies of the same pathogen according to the present application;

[0025] Figure 3 The structure diagram of the cover sheet (perspective) according to the present application;

[0026] Figure 4 The structure diagram of the substrate according to the present application;

[0027] Figure 5 The cross-sectional view of the immunomicrofluidic chip for the combined detection of IgM & IgG antibodies of the same pathogen according to the present application;

[0028] Figure 6 The diagram of the liquid flow state in the microchannel according to the present application;

[0029] Figure 7 The principle diagram of the microfluidic chip based on laminar flow for dropping the sample to be detected according to the present application;

[0030] Figure 8 The principle diagram of the microfluidic chip based on laminar flow for dropping buffer according to the present application;

[0031] Figure 9 The principle diagram of the microfluidic chip for the combined detection of IgM & IgG antibodies of the same pathogen for dropping the sample to be detected according to the present application;

[0032] Figure 10 The principle diagram of the sample to be detected on the chip according to the present application in the reaction stage; Figure 9

[0033] The principle diagram of the sample to be detected on the chip according to the present application in the reaction stage; Figure 11 Figure 10 The principle diagram of the sample to be detected on the chip according to the present application for absorbing waste liquid through the magnetic control intercept valve;

[0034] Figure 12 Figure 11 The principle diagram of the chip according to the present application for dropping buffer;

[0035] Figure 13 The structure diagram of the top layer of the microfluidic chip based on laminar flow for verifying the distribution of biological molecules according to the present application;

[0036] Figure 14 The structure diagram of the bottom layer of the microfluidic chip based on laminar flow for verifying the distribution of biological molecules according to the present application;

[0037] Figure 15 The structure diagram of the microfluidic chip for the combined detection of IgM & IgG antibodies of the same pathogen according to the present application in one embodiment.

[0038] ​​In the figure, 1 is a substrate; 2 is a cover sheet; 3 is a microchannel; 4 is a buffer injection hole; 5 is a liquid flow regulating valve; 6 is a sample injection hole; 7 is a magnetic control intercept valve; 31 is a marker area; 32 is an IgG detection area; and 33 is an IgM detection area. DETAILED DESCRIPTION

[0039] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0040] Unless defined, the technical terms used in the following embodiments have the same meanings as generally understood by those skilled in the art to which the present application belongs. The test reagents used in the following embodiments have the same meanings as generally understood by those skilled in the art to which the present application belongs, unless otherwise specified. The experimental methods used in the following embodiments have the same meanings as generally understood by those skilled in the art to which the present application belongs, unless otherwise specified.

[0041] The present application relates generally to the field of immunodetection technology, and relates to an immunomicrofluidic chip for combined detection of IgM and IgG antibodies of the same pathogen and application thereof. In order to solve the bottleneck problem of mutual influence between IgM and IgG antibodies in the existing combined detection technology of pathogen antibodies, a layered microfluidic chip is proposed, which uses capture method to detect specific IgM antibody and uses indirect method to detect specific IgG antibody. The IgM antibody capture detection area and the IgG antibody indirect detection area are integrated in the independent fluid layer of the chip, respectively, so that after injection of a single sample, the detection of IgM and IgG antibodies in the same channel can be realized synchronously but without interference.

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

[0043] As Figures 2-5As shown, the application provides an immune microfluidic chip for combined detection of IgM and IgG antibodies of the same pathogen, comprising a substrate 1 and a cover sheet 2 pressed on the substrate 1, the substrate 1 and the cover sheet 2 enclosing a microchannel 3, the height of the microchannel 3 being 20-50 μm, the left end of the microchannel 3 being in communication with a buffer injection hole 4 opened on the cover sheet 2, the right end of the microchannel 3 being provided with a liquid flow control valve 5, the liquid flow control valve 5 controlling the opening and closing of the flow path of the liquid in the microchannel 3 to the liquid flow control valve 5, the microchannel 3 being provided with a labeling area 31 and a detection area from left to right, the detection area comprising an IgG detection area 32 and an IgM detection area 33, the cover sheet 2 being further provided with a sample injection hole 6, the sample injection hole 6 being arranged between the detection area and the liquid flow control valve 5, the microchannel 3 being further provided with a magnetic control interception valve 7, the magnetic control interception valve 7 being arranged between the labeling area 31 and the detection area, the magnetic control interception valve 7 being arranged to control the opening and closing of the flow path between the labeling area 31 and the detection area in the microchannel 3 by magnetic attraction, the upper surface (chip bottom layer) of the substrate 1 being coated with labeled anti-human IgG antibody at the position corresponding to the labeling area 31, the upper surface (chip bottom layer) of the substrate 1 being coated with pathogen recombinant antigen at the position corresponding to the IgG detection area 32, the lower surface (chip top layer) of the cover sheet 2 being coated with labeled antigen at the position corresponding to the labeling area 31, the lower surface (chip top layer) of the cover sheet 2 being coated with anti-human IgM antibody at the position corresponding to the IgM detection area 32, the IgG detection area 31 and the IgM detection area 32 not coinciding in the vertical plane.

[0044] In the formula, the specific IgM antibody is detected by capture method, the specific IgG antibody is detected by indirect method, and the IgM antibody capture detection area and the IgG antibody indirect detection area are integrated in the independent fluid layer of the chip, and the height of the microchannel is accurately controlled to maintain the liquid flow in a stable laminar flow state, so that the detection of IgM and IgG antibodies in the same channel can be realized after a single sample injection, that is, the pathogen specific IgM antibody in the upper layer of the liquid flow is specifically captured by the anti-human IgM antibody pre-coated on the top layer of the chip and combined with the labeled antigen, the pathogen specific IgG antibody in the lower layer of the liquid flow is specifically captured by the recombinant antigen pre-coated on the bottom layer of the chip and combined with the labeled anti-human IgG antibody, the space separation between the IgM antibody and IgG antibody detection areas eliminates the risk of non-target antibody combination, effectively avoiding the mutual influence problem in the traditional detection of pathogen specific IgM / IgG antibody combined detection.

[0045] In fluid mechanics, the Reynolds number (Re) is a key parameter for judging the flow state of a liquid. When Re is lower than the critical value, the flow is laminar. In a microfluidic channel, Re<100 can be considered as laminar flow.

[0046] According to the calculation formula of Reynolds number: Re = (ρ is the fluid density, v is the average flow velocity, Dh is the hydraulic diameter, μ is the fluid dynamic viscosity), for rectangular microchannels, Dh = 4wh (w is the channel width, h is the channel height), since the width of the microchannel is much greater than the height, Dh can be simplified as Dh = 2h, so the height of the microchannel is the key parameter to determine the flow state of the liquid in the microchannel.

[0047] As shown in Figure 6 , for common fluids (such as water, buffer), at a typical flow rate (0.1-10 mm / s), the channel height h is usually set to 10-100 μm to ensure that Re < 100. For other liquids with faster flow rates, if the microchannel height is not properly designed, the internal liquid flow will exhibit irregular turbulent flow, affecting the reactions at different levels. Therefore, limiting the microchannel height to 20-50 μm can ensure that any liquid flow in the microchannel exhibits a smooth laminar flow state.

[0048] The working procedure of the layered microfluidic chip is as follows:

[0049] Molecule capture stage: as shown in Figure 7 , the liquid flow control valve 5 is turned to the far right, so that it is not connected to the microchannel 3. After adding the sample to be tested to the sample injection hole, the sample flows to the left and right. Since the microchannel 3 is not connected to the liquid flow control valve 5, the liquid flowing to the right is stopped at the end of the microchannel 3 under the action of surface tension and does not flow out spontaneously, while the liquid flowing to the left can continue to flow to the detection area, where the detected substance binds to the solidified antigen / antibody in the detection area; the liquid flow continues to flow to the magnetic control interception valve 7 and is intercepted there. Since the water absorption provided by the magnetic control interception valve 7 is greater than the capillary force in the microchannel, the liquid flow is immediately collected into the magnetic control interception valve and does not continue to flow to the left. After the liquid flow in the microchannel is completely collected into the magnetic control interception valve 7, the interception valve is removed, and the microchannel is restored to be unobstructed to ensure that the subsequent reagent flow and reaction are not disturbed.

[0050] B. Label substance binding stage: as shown in Figure 8 , the liquid flow control valve is turned to the far left, so that it is embedded in the microchannel. Add buffer solution to the buffer injection hole, which enters the microchannel under the action of capillary driving force, dissolves the label substance in the labeling area, and the remaining liquid flow is collected into the liquid flow control valve.

[0051] The IgG detection area and the IgM detection area are arranged in order along the microchannel from left to right.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] The flow control valve 5 is made of a movable absorbent material, which moves to contact or move away from the microchannel 3.

[0057] The principle of combined detection of pathogen-specific IgM and IgG is as follows.

[0058] Detection molecule capture stage:

[0059] 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, under the action of the surface tension at the end, the liquid flowing to the right stays at the end of the microchannel 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 (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 11As 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.

[0060] Labeling process:

[0061] 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.

[0062] 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.

[0063] Example 1: In-Channel Laminar Flow Verification of a Chip

[0064] 1. Chip fabrication

[0065] 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.

[0066] 2. Biomolecular coating procedures

[0067] First, 1.5 μL streptavidin was spotted on the top layer or bottom layer of the chip at the position corresponding to the IgM detection zone, incubated for 1 h and washed, and then dried. After that, biotinylated anti-human IgM antibody was spotted on the IgM detection zone, and incubation was continued for 1 h. After washing again, fluorescent microspheres coupled with MP recombinant antigen were spotted on the top layer or bottom layer of the chip at the position corresponding to the labeling zone, and dried at 37°C.

[0068] 3. Assembly procedure of the microfluidic chip

[0069] First, the magnetic control intercept valve was embedded in the liquid guide hole of the top layer of the chip; second, the liquid flow control valve was placed in the waste chamber of the top layer of the chip; finally, according to the purpose of verification, the top layer and the bottom layer of the chip were assembled and bonded (i.e., in the experimental group, the labeling zone and the detection zone were located on the same level, and in the control group, the labeling zone and the detection zone were located on different levels).

[0070] The distribution of biomolecules in the top layer of the chip verified by laminar flow is shown in Table 1-1, and the specific structure is shown in Figure 13 , wherein A is the experimental group, and B is the control group.

[0071] Table 1-1 Verification of the top layer of the chip by laminar flow

[0072]

[0073] The distribution of biomolecules in the bottom layer of the chip verified by laminar flow is shown in Table 1-2, and the specific structure is shown in Figure 14 , wherein A is the experimental group, and B is the control group.

[0074] Table 1-2 Verification of the bottom layer of the chip by laminar flow

[0075]

[0076] 4. Detection procedure

[0077] First, the liquid flow control valve is set to the rightmost position, so that it is not connected to the microchannel. 10 μL of sample is added to the sample injection hole. The sample flows to the left and right. Since the microchannel is not connected to the liquid flow control valve, the liquid flowing to the right is stopped at the end of the microchannel due to the surface tension, and does not flow out spontaneously. The liquid flowing to the left continues to flow until it reaches the detection zone, where the MP-specific IgM antibody in the sample is captured by the anti-human IgM antibody in the detection zone. The liquid flow is then intercepted by the magnetic control valve, and is collected in the magnetic control valve due to the greater water absorption force provided by the magnetic control valve than the capillary force in the microchannel. Second, after the liquid flow in the microchannel is completely collected in the magnetic control valve, the magnetic control valve is removed, and the microchannel is restored to be unobstructed, so that the subsequent reagent flow and reaction are not disturbed. Third, the liquid flow control valve is set to the leftmost position, so that it is embedded in the microchannel. Buffer is added to the buffer injection hole, and enters the microchannel under the action of capillary force. When the buffer flows through the labeling zone, the fluorescent microspheres coupled with MP recombinant antigen are dissolved, and the labeling substance reaches the detection zone and binds to the specific IgM antibody captured in the detection zone. The remaining liquid flow is collected in the liquid flow control valve. Finally, the fluorescence signal value of the detection zone is read by the analyzer.

[0078] 5. Experimental results

[0079] Table 2-1 Top layer verification results of the chip

[0080]

[0081] Table 2-2 Bottom layer verification results of the chip

[0082]

[0083] According to the experimental data, only when the labeling zone and the detection zone are arranged on the same layer, the detection zone can detect the signal of the test substance. When the labeling zone and the detection zone are arranged on different layers, the detection zone cannot capture the labeling substance, and the detection zone does not detect the signal of the test substance, which confirms that the liquid flow in the microchannel is in a laminar flow state.

[0084] Example 2: Combined detection of MP-IgM and IgG antibodies

[0085] 1. Chip manufacturing

[0086] The top layer and the bottom layer of the chip are both made of PMMA. The microchannel structure, the sample injection hole, the buffer injection hole, the flow guide hole and other structures of the top layer are designed by CAD software, and then processed by a CO2 laser etching machine.

[0087] 2. Bionmolecule coating procedure of the top layer of the chip

[0088] First, 1.5 μL of streptavidin was spotted on the top layer of the chip at the position corresponding to the IgM antibody detection area, incubated for 1 h and washed, and after drying, biotinylated anti-human IgM antibody was spotted on the IgM antibody detection area, and incubation was continued for 1 h. After washing again, fluorescent microsphere-coupled MP recombinant antigen was spotted at the position of the labeling area, and drying was carried out at 37°C.

[0089] 3. Biomolecule coating procedure of the bottom layer of the chip

[0090] First, 1.5 μL of streptavidin was spotted on the bottom layer of the chip at the position corresponding to the IgG antibody detection area, incubated for 1 h and washed, and after drying, biotinylated MP recombinant antigen was spotted on the IgG antibody detection area, and incubation was continued for 1 h. After washing again, fluorescent microsphere-coupled anti-human IgG antibody was spotted at the position of the labeling area, and drying was carried out at 37°C.

[0091] Note: For the traditional bidirectional microfluidic chip, the IgM antibody detection area and the IgG antibody detection area are both arranged on the bottom layer of the chip, and the biomolecule coating procedure is the same as above.

[0092] 4. Assembly procedure of the microfluidic chip

[0093] First, the magnetic control intercept valve was embedded in the liquid guide hole of the top layer of the chip; second, the liquid flow control valve was placed in the waste liquid cavity of the top layer of the chip; and finally, the top layer and the bottom layer of the chip were tightly attached and bonded.

[0094] 5. Detection procedure

[0095] Two samples S1 and S2 were selected and detected by the conventional bidirectional flow microfluidic chip and the double-layer microfluidic chip of the present application, respectively. The detection of the conventional bidirectional flow microfluidic chip was as described in CN221926380U, and the detection procedure of the double-layer microfluidic chip of the present application was the same as in Example 1.

[0096] 6. Experimental results

[0097] Table 3 Detection results of MP-IgM and IgG

[0098]

[0099] 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.

[0100] 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.

[0101] Example 3: Combined detection of IgM and IgG antibodies against Chikungunya and Dengue viruses

[0102] 1. Chip Structure

[0103] 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).

[0104] 2. Chip fabrication

[0105] 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.

[0106] 3. Biomolecular coating program on the top layer of the chip

[0107] First, 1.5 μL streptavidin was spotted in T2 and T4 detection areas of the top layer of the chip successively, incubated for 1 h and washed, dried, and then biotinylated anti-human IgM antibody was spotted in the T2 detection area and biotinylated dengue recombinant antigen was spotted in the T4 detection area, and incubation was continued for 1 h. After washing again, fluorescent microsphere-coupled CHIKV recombinant antigen and fluorescent microsphere-coupled anti-human IgG antibody were spotted at the position of the labeling area, and drying was carried out at 37℃.

[0108] 4. Biomolecule coating procedure of the bottom layer of the chip

[0109] First, 1.5 μL streptavidin was spotted in T2 and T4 detection areas of the top layer of the chip successively, incubated for 1 h and washed, dried, and then biotinylated anti-human IgM antibody was spotted in the T2 detection area and biotinylated dengue recombinant antigen was spotted in the T4 detection area, and incubation was continued for 1 h. After washing again, fluorescent microsphere-coupled CHIKV recombinant antigen and fluorescent microsphere-coupled anti-human IgG antibody were spotted at the position of the labeling area, and drying was carried out at 37℃.

[0110] 5. Assembly procedure of the microfluidic chip

[0111] First, the magnetic control intercept valve was embedded into the liquid guide hole of the top layer of the chip; second, the liquid flow control valve was placed in the waste liquid cavity of the top layer of the chip; and finally, the top layer and the bottom layer of the chip were tightly attached and bonded.

[0112] 6. Detection procedure

[0113] Three different samples S1, S2 and S3 were selected for detection, and the detection steps were the same as in Example 1.

[0114] 7. Experimental results

[0115] Table 4: Detection results of clinical samples

[0116]

[0117] The above technical solutions only reflect the preferred technical solutions of the present application, and some changes that can be made by those skilled in the art to some parts thereof all embody the principles of the present application and are within the protection scope of the present application.

[0118] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0119] 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.

[0120] 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 combined detection of IgM and IgG antibodies to the same pathogen, comprising a substrate and a cover plate pressed on the substrate, the substrate and the cover plate enclosing a microchannel, characterized in that, The height of the microchannel is 20-50 μm, the left end of the microchannel is communicated with the buffer injection hole opened on the cover sheet, 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, the microchannel is sequentially provided with a marking area and a detection area from left to right, the detection area includes an IgG detection area and an IgM detection area, the cover sheet is further provided with a sample injection hole, the sample injection hole is arranged between the detection area and the liquid flow control valve, a magnetic control intercept valve is arranged in the microchannel, the magnetic control intercept valve is arranged between the marking area and the detection area, 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 by magnet adsorption, the upper surface of the substrate is coated with a labeled anti-human IgG antibody at a position corresponding to the marking area, the upper surface of the substrate is coated with a pathogen recombinant antigen at a position corresponding to the IgG detection area, the lower surface of the cover sheet is coated with a labeled antigen at a position corresponding to the marking area, the lower surface of the cover sheet is coated with an anti-human IgM antibody at a position corresponding to the IgM detection area, the IgG detection area and the IgM detection area do not coincide in the vertical plane, the cover sheet is provided with a flow guide hole, the bottom of the magnetic control intercept valve passes through the flow guide hole and contacts the upper surface of the substrate, the top of the magnetic control intercept valve is provided with a 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 magnetic force, the bottom of the magnetic control intercept valve is provided with a 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 immunomicrofluidic chip for simultaneous detection of IgM & IgG antibodies to the same pathogen according to claim 1, characterized in that, The IgG detection area and the IgM detection area are sequentially arranged along the microchannel from left to right.

3. The immunomicrofluidic chip for simultaneous detection of IgM and IgG antibodies to 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 along the microchannel from left to right, and the IgG detection area and the IgM detection area of each group correspond to detect specific IgM and IgG antibodies of the same pathogen.

4. The immunomicrofluidic chip for simultaneous detection of IgM & IgG antibodies to the same pathogen according to claim 1, wherein, The lower surface of the cover sheet is provided with a groove along the length direction, the cover sheet and the upper surface of the substrate form a microchannel through the groove, and the width of the microchannel is 2-3 mm.

5. The immunomicrofluidic chip for simultaneous detection of IgM & IgG antibodies to the same pathogen according to claim 2, wherein, The horizontal distance from the IgG detection area to the buffer injection hole is 30-35 mm, and the horizontal distance from the IgM detection area to the buffer injection hole is 40-45 mm.

6. The immunomicrofluidic chip for simultaneous detection of IgM & IgG antibodies to the same pathogen according to claim 1, wherein, The liquid flow control valve is a movable water-absorbing material, and the water-absorbing material moves to contact or away from the microchannel.

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