Microfluidic chips with increased detection sites based on dual-color and dual-layer technologies and their applications

Abstract

This invention proposes a microfluidic chip and its application based on dual-color and dual-layer technology to increase detection sites. The chip includes a substrate and a cover plate laminated onto the substrate. The substrate and cover plate enclose a microchannel. The microchannel is provided with a labeling region and a detection region. The labeling region includes a first labeling region on the lower surface of the cover plate and a second labeling region on the upper surface of the substrate. The detection region includes multiple detection sites on the lower surface of the cover plate and the upper surface of the substrate. The first labeling region is coated with a first labeled antigen / antibody, and the second labeling region is coated with a second labeled antigen / antibody. The first and second labeled antigens / antibodies are fluorescent microspheres labeled with two fluorophores that have the same excitation spectrum but different emission spectra. The multiple detection sites are respectively coated with capture antigens / antibodies that bind to different target analytes. By using a layered design combined with dual-color labeling, the number of simultaneously detectable indicators within the microchannel is increased.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostics and immunoassay technology, and specifically relates to a microfluidic chip based on dual-color and dual-layer technology to increase detection sites and its application. Background Technology

[0002] Microfluidics technology refers to the technique of processing or manipulating microfluidics using microchannels ranging from tens to hundreds of micrometers. It integrates a series of complex operations required for detection and analysis, such as sample pretreatment, sample addition, reaction, separation, and detection, into a microfluidic chip of just a few square centimeters. Furthermore, it provides the sealed environment needed for in vitro diagnostic testing, enabling low-consumption, rapid, and efficient sample analysis. Its greatest advantage lies in the flexible combination of various functional units, achieving not only low reagent consumption and multi-indicator simultaneous detection, but also significant advantages such as ease of integration and good compatibility with other technologies and equipment.

[0003] Immunomicrofluidics combines immunoassay techniques with microfluidics, enabling specific immune reactions to proceed sequentially on a controlled microfluidic platform. Compared to traditional detection platforms, this technology integrates the antigen-antibody reaction process onto the chip surface. This micro- and nano-scale fluid manipulation and integration not only improves the reaction speed of antigens and antibodies but also significantly shortens reaction time, greatly simplifying the immunoassay process.

[0004] Lateral flow immunoassay microfluidics is an emerging point-of-care testing platform that integrates lateral flow immunoassay and microfluidics. Its core lies in using micrometer-level channel structures to replace traditional nitrocellulose chromatography membranes. The antigen-antibody reaction takes place within these micrometer-level channels, which are formed by a substrate and a cover plate. One end of the microchannel connects to a sample well on the cover plate, while the other end connects to a waste collection tank. Within the microchannel, labeled areas and 1-6 detection points are sequentially arranged along the sample flow direction. The labeled areas on the substrate are pre-dried and labeled with antibodies or antigens, and multiple detection areas achieve indirect coating of antigens or antibodies through a biotin-avidin system.

[0005] In clinical practice, the need for multi-indicator combined detection methods is increasingly urgent. For example, in the initial screening of allergens, it is necessary to systematically screen patients for dozens of common allergens to identify the specific allergens causing allergic reactions, providing a basis for personalized treatment. Furthermore, the detection of cytokines (such as IL-6, TNF-α, and IFN-γ) can reflect the activity status of the immune system, which is of great significance for the diagnosis and prognostic assessment of inflammatory responses, tumor immunity, and various immune-related diseases. In addition, in the diagnosis of infectious diseases, the combined detection of inflammatory markers and multiple pathogens helps to assess the degree of infection and identify the type of pathogen. Therefore, multi-indicator combined detection not only improves the efficiency of clinical diagnosis but also provides doctors with more comprehensive information, thereby enabling the development of more precise treatment plans.

[0006] However, methods such as chemiluminescence often involve single-index detection followed by combined analysis. This approach is not only inefficient and consumes a lot of samples, but also suffers from significant drawbacks such as accumulated operational errors and high costs. In contrast, lateral flow immunomicrofluidic technology, with its advantages of miniaturization and integration, can achieve simultaneous detection of multiple target analytes under single sample injection conditions. This effectively overcomes the limitations of traditional methods, significantly shortens reporting time, and reduces sample volume and reagent consumption, providing a powerful technical platform to meet the clinical demand for efficient, accurate, and portable multi-index combined testing.

[0007] In existing lateral flow immunomicrofluidic chips, the detection region is typically 30 mm long, and the area of ​​a single detection point is 1 mm². 2 To avoid interference between fluorescence signals at detection points, the distance between two detection points must be ≥5mm. Therefore, due to space limitations, this type of chip can only simultaneously detect a maximum of 6 indicators. To increase the number of detection indicators, the overall microchannel length must be extended. However, this brings the following problems: ① Increased manufacturing cost: Extending the microchannel length requires remaking the chip mold, which is costly; ② Non-linear decrease in flow rate: According to the Hagen-Poiseuille law, the flow rate of liquid in the microchannel is inversely proportional to the length of the microchannel. As the microchannel length increases, the flow rate in the channel decreases non-linearly. In multi-indicator detection, the flow rate difference caused by the long channel will cause asynchronous reaction times at each detection point. For example, indicators near the end of the channel will have shorter reaction times than those at the front, affecting detection consistency; ③ Risk of flow stagnation: When the microchannel length exceeds the capillary action threshold, capillary flow interruption may occur. Summary of the Invention

[0008] This invention aims to provide a microfluidic chip and its application based on dual-color and dual-layer technology to increase detection sites. By optimizing the chip design and detection method, it solves the problems existing in the prior art and proposes a laminar flow-based microfluidic chip. The top and bottom layers of the chip can be regarded as two independent fluid reaction layers. By selecting two fluorescent microsphere probes with the same excitation spectrum but different emission spectra, the number of detection sites can be doubled in the same physical space without mutual interference, ultimately achieving double the detection of indicators and improving detection efficiency.

[0009] To achieve the above objectives, the technical solution of the present invention is as follows: A microfluidic chip with increased detection sites based on dual-color and dual-layer technology includes a substrate and a cover plate pressed onto the substrate. The substrate and the cover plate enclose a microchannel with a height of 20-50 μm. The left end of the microchannel communicates with a buffer injection hole on the cover plate, and a flow control valve is provided at the right end of the microchannel. The flow control valve controls the opening and closing of the flow path of liquid in the microchannel to the flow control valve. From left to right, the microchannel is provided with a marking area and a detection area. The marking area includes a first marking area on the lower surface of the cover plate and a second marking area on the upper surface of the substrate. The detection area includes multiple detection sites on the lower surface of the cover plate and multiple detection sites on the upper surface of the substrate. The detection sites on the lower surface of the cover plate and the upper surface of the substrate do not overlap in the vertical plane. The cover plate also has... A sample injection port is provided, which is located between the detection zone and the flow control valve. A magnetically controlled intercept valve is provided between the labeling zone and the detection zone of the microchannel. The magnetically controlled intercept valve uses a magnet to attract and control the opening and closing of the flow path between the labeling zone and the detection zone in the microchannel. The first labeling zone is coated with a first labeled antigen / antibody, and the second labeling zone is coated with a second labeled antigen / antibody. The first labeled antigen / antibody and the second labeled antigen / antibody are fluorescent microspheres labeled with two fluorophores that have the same excitation spectrum but different emission spectra. The multiple detection sites are respectively coated with capture antigens / antibodies that bind to different target analytes.

[0010] In one embodiment of the present invention, the first labeled antigen / antibody is labeled with FITC fluorescent microspheres, and the second labeled antigen / antibody is labeled with PE fluorescent microspheres.

[0011] In one embodiment of the present invention, a groove is provided on the lower surface of the cover sheet along its length direction, and the cover sheet forms a microchannel by means of the groove and the upper surface of the substrate, wherein the width of the microchannel is 2-3mm.

[0012] In one embodiment of the present invention, the length of the detection area is 30 mm, and the distance between two adjacent detection sites on the same horizontal plane is greater than or equal to 5 mm.

[0013] In one embodiment of the present invention, the distance between two adjacent detection sites on the same horizontal plane is the same.

[0014] In one embodiment of the present invention, the number of detection sites on the lower surface of the cover sheet is less than or equal to 6, and the number of detection sites on the upper surface of the substrate is less than or equal to 6.

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

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

[0017] On the other hand, the present invention also provides an application for multi-index detection using the microfluidic chip in any of the above technical solutions, comprising at least the following steps:

[0018] 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. When it reaches the detection area, the test substance in the sample comes into contact with the captured antigen / antibody on the upper surface of the substrate and the lower surface of the cover, forming an antigen-antibody complex. When the liquid continues to flow and reaches the magnetic intercept valve, it is intercepted and collected into the magnetic intercept valve. After all the liquid in the channel has been collected into the magnetic intercept valve, remove the intercept valve. At this time, the microchannel is restored to an unobstructed state.

[0019] 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, dissolving the labeled antigens / antibodies on the upper surface of the substrate and the labeled areas under the cover, respectively, and binding to the complex molecules of different detection sites on the top and bottom layers of the chip. The remaining liquid flow is collected into the flow control valve.

[0020] 3) After the reaction is complete, the substrate is excited by an excitation light source. First, the fluorescence signals of different detection sites on the underside of the cover are collected using the emission spectrum corresponding to the fluorescein labeled on the fluorescent microspheres in the first labeled region. Then, the substrate is excited again, and the fluorescence signals of different detection sites on the upper surface of the substrate are collected using the emission spectrum corresponding to the fluorescein labeled on the fluorescent microspheres in the second labeled region.

[0021] The microfluidic chip obtained through the above technical solution, which increases detection sites based on dual-color and dual-layer technology, and its application, have the following beneficial effects:

[0022] 1. Layered design to increase the number of synchronous detection indicators in the microchannel: By treating the top and bottom layers of the chip as two independent fluid reaction layers, six detection sites are designed on each of the top and bottom layers. In the same physical space, the number of detection sites is doubled (12), which improves detection efficiency and reduces manufacturing costs.

[0023] 2. Dual-color labeling eliminates signal interference: The top and bottom layers of the chip are labeled with two different fluorescent microsphere probes that have the same excitation spectrum but different emission spectra. This ensures that the detection of the top layer is not affected by the fluorescence signal of the adjacent bottom layer, and vice versa. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the microfluidic chip (transparent) based on dual-color and dual-layer technology to increase detection sites as described in this invention;

[0025] Figure 2 This is a schematic diagram of the cover plate (transparent) of the present invention;

[0026] Figure 3 This is a schematic diagram of the structure of the substrate described in this invention;

[0027] Figure 4 This is a cross-sectional view of the microfluidic chip with increased detection sites based on dual-color and dual-layer technology described in this invention;

[0028] Figure 5 This is a schematic diagram illustrating the principle of adding a sample to be tested using a microfluidic chip based on dual-color and dual-layer technology to increase detection sites, as described in this invention.

[0029] Figure 6 This is a schematic diagram of the microfluidic chip for adding buffer solution based on dual-color and dual-layer technology to increase detection sites, as described in this invention.

[0030] Figure 7 This is a distribution map of detection sites for sIgE antibody detection of inhaled and ingested allergens using the microfluidic chip described in this invention.

[0031] 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; 31a is the first labeling area; 31b is the second labeling area; 32 is the detection area; and 32a is the detection site. Detailed Implementation

[0032] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0033] 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.

[0034] This invention relates to the field of immunoassay technology, specifically to a microfluidic chip that increases detection sites based on dual-color and dual-layer technology. The chip's top and bottom layers are treated as two independent fluid reaction layers. Two fluorescent microsphere probes with the same excitation spectrum but different emission spectra (such as FITC and FE fluorescent microspheres, both with an excitation wavelength of 488 nm, but FITC with an emission wavelength of 525 nm and FE with an emission wavelength of 575 nm) are placed on the top and bottom layers of the chip, respectively, at positions corresponding to the labeled areas. Multiple detection sites are simultaneously established on both the top and bottom layers, increasing detection indicators within a limited length of microchannel, while ensuring that the detection sites on the top and bottom layers do not interfere with each other.

[0035] 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.

[0036] like Figure 1-4As shown, this invention proposes a microfluidic chip with increased detection sites based on dual-color and dual-layer technology, including a substrate 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 communicates with a buffer injection hole 4 on the cover plate 2, and the right end of the microchannel 3 is provided with a flow control valve 5. The flow control valve 5 controls the opening and closing of the flow path of liquid in the microchannel 3 to the flow control valve 5. From left to right, the microchannel 3 is provided with a marking area 31 and a detection area 32. The marking area 31 includes a first marking area 31a on the lower surface of the cover plate 2 and a second marking area 31b on the upper surface of the substrate 1. The detection area 32 includes multiple detection sites 32a on the lower surface of the cover plate 2 and multiple detection sites 32a on the upper surface of the substrate 1. The detection sites 32a on the lower surface of the cover plate 2 and the upper surface of the substrate 1 do not overlap in the vertical plane. The cover plate 2 is also provided with a sample injection hole 6, which is located between the detection area 32 and the flow control valve 5. A magnetically controlled intercept valve 7 is provided between the labeling area 31 and the detection area 32 of the microchannel 3. The magnetically controlled intercept valve 7 uses a magnet to attract and control the opening and closing of the flow path between the labeling area 31 and the detection area 32 in the microchannel 3. The first labeling area 31a is coated with a first labeled antigen / antibody, and the second labeling area 31b is coated with a second labeled antigen / antibody. The first labeled antigen / antibody and the second labeled antigen / antibody are fluorescent microspheres labeled with two fluoresceins with the same excitation spectrum but different emission spectra, respectively. The multiple detection sites 32a are respectively coated with capture antigens / antibodies that bind to different target analytes.

[0037] 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.

[0038] 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.

[0039] For common fluids (such as water and buffer solutions), at typical flow rates (0.1-10 mm / s), the channel height h is usually set to 10-100 μm to ensure Re < 100. However, for other liquids with faster flow rates, if the microchannel height is not designed properly, the internal flow will exhibit an irregular turbulent state, causing reactions at different levels to interfere with each other. Therefore, limiting the microchannel height to 20-50 μm ensures that any liquid flow exhibits a stable laminar flow state within the microchannel.

[0040] Therefore, in lateral flow microchannels, when Re < 100, the liquid flow is in a laminar state. With this characteristic, the top and bottom layers of the chip can be regarded as two independent fluid reaction layers.

[0041] In a laminar flow-based microfluidic chip setup, two fluorescent microsphere probes with the same excitation spectrum but different emission spectra (such as FITC fluorescent microspheres and PE fluorescent microspheres, both with an excitation wavelength of 488 nm, where FITC has an emission wavelength of 525 nm and PE has an emission wavelength of 575 nm) are selected and placed at the corresponding marking areas on the lower surface of the cover (top layer of the chip) and the upper surface of the substrate (bottom layer of the chip), respectively. At the same time, multiple detection sites (such as 12 detection sites, including 6 detection sites on the top layer of the chip and 6 detection sites on the bottom layer of the chip) are uniformly distributed along the detection area in the direction of liquid flow within the microchannel, with the detection sites on the top and bottom layers being staggered.

[0042] When initiating bidirectional immunomicrofluidic detection, firstly, the capture molecules (capture antigens or antibodies) located in the detection area bind to the target molecules in the sample to form antigen-antibody complexes; secondly, the reverse lateral flow is initiated to dissolve the labeled antigens or antibodies in the top and bottom labeling areas of the chip, and bind to the complex molecules at different detection sites in the top and bottom layers of the chip.

[0043] After the reaction, the chip is excited using an excitation light source. First, the fluorescence signals from different detection sites on the top layer of the chip are collected using the emission spectrum corresponding to the fluorescein labeled on the lower surface of the cover plate (top layer of the chip). Then, the chip is excited again, and the fluorescence signals from different detection sites on the bottom layer of the chip are collected using the emission spectrum corresponding to the fluorescein labeled on the upper surface of the substrate (bottom layer of the chip). Because the emission spectra of the top and bottom layers of the chip are different, the detection of the top layer of the chip is not interfered with by the fluorescence signals of the adjacent bottom layer of the chip; similarly, the detection of the bottom layer of the chip is also not interfered with by the fluorescence signals of the adjacent top layer of the chip.

[0044] The first labeled antigen / antibody is labeled with FITC fluorescent microspheres, and the second labeled antigen / antibody is labeled with PE fluorescent microspheres.

[0045] The cover plate 2 has a groove along its length on its lower surface. The cover plate 2 and the upper surface of the substrate 1 are enclosed by the groove to form a microchannel 3. The width of the microchannel 3 is 2-3 mm.

[0046] The length of the detection area 32 is 30 mm, and the distance between two adjacent detection sites 32a on the same horizontal plane (upper surface of the substrate or lower surface of the cover plate) is greater than or equal to 5 mm.

[0047] The distance between two adjacent detection sites 32a on the same horizontal plane is the same.

[0048] The number of detection sites 32a on the lower surface of the cover plate 2 is less than or equal to 6, and the number of detection sites 32a on the upper surface of the substrate 1 is less than or equal to 6.

[0049] The cover plate 1 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.

[0050] The flow control valve 5 is made of a movable absorbent material, which moves to contact or move away from the microchannel.

[0051] like Figure 4 As shown, the distribution structure of the laminar flow microfluidic chip based on dual-color labeling for multi-index detection is as follows:

[0052] Cover sheet: From left to right, it consists of a buffer injection port, a first labeling area, a magnetically controlled intercept valve, a detection area, a sample injection port, and a flow control valve (made of absorbent material).

[0053] Substrate: From left to right, these are the second marker area and the detection area.

[0054] The first and second labeled regions each contain a fluorescent microsphere probe with the same excitation spectrum but different emission spectra. Furthermore, 12 detection sites are uniformly dispersed along the liquid flow direction within the microchannel, including 6 detection sites on the lower surface of the cover and 6 detection sites on the upper surface of the substrate, with the detection sites on the top and bottom layers being staggered.

[0055] The specific workflow is as follows:

[0056] A. Detection molecule capture stage: such as Figure 5As shown, the flow control valve is switched to the far right, disconnecting it from the microchannel. After adding 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 does not flow out spontaneously, while the liquid flowing to the left continues to flow. When it reaches the detection area, the analyte in the sample comes into contact with the capture molecules at the detection sites on the top and bottom layers of the chip, forming antigen-antibody complexes. 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 does not continue to flow to the left. After all the liquid in the microchannel has been collected into the magnetically controlled intercept valve, the intercept valve is removed, and the microchannel is restored to its original state, ensuring that subsequent reagent flow and reactions are not disturbed.

[0057] B. The stage of marker substance binding: such as Figure 6 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 well. Under the action of capillary driving force, the buffer solution enters the microchannel and dissolves the labeled antigens or antibodies in the top and bottom labeling regions of the chip, respectively. The buffer solution binds to the complex molecules at different detection sites in the top and bottom layers of the chip. The remaining liquid flow is collected in the flow control valve.

[0058] C. Excitation and Detection Stage: After the reaction, the chip is excited using an excitation light source. First, fluorescence signals at different detection sites are collected using the emission spectrum corresponding to the fluorescein labeled on the top layer of the chip. Second, the chip is excited again, and fluorescence signals at different detection sites are collected using the emission spectrum corresponding to the fluorescein labeled on the bottom layer of the chip. Because the emission spectra of the top and bottom layers of the chip are different, the detection of the top layer is not interfered with by the fluorescence signals of the adjacent bottom layer; similarly, the detection of the bottom layer is also not interfered with by the fluorescence signals of the adjacent top layer.

[0059] Example: Detection of sIgE antibodies against inhaled and ingested allergens

[0060] 1. Chip fabrication

[0061] Both the substrate and cover plate were fabricated using PMMA material. The microchannel structure, sample injection port, buffer injection port, liquid guiding port, and other structures of the cover plate were designed using CAD software, and then the PMMA surface was processed using a CO2 laser etching machine. Finally, the microchannel surface of the chip was treated with a hydrophilic coating.

[0062] 2. Biomolecular coating procedures

[0063] Chip top-level biomolecular coating program: such as Figure 7As shown, firstly, 1.5 μL of streptavidin was sequentially spotted at the detection sites (T1, T3, T5, T7, T9, T11) on the top layer of the chip, incubated for 1 h, washed, and dried. Afterward, biotin-labeled inhaled allergens (house dust mite, dust mites, artemisia, ragweed, hops, and mold) were spotted at the same detection sites, and incubated for another 1 h. After another wash, FITC fluorescent microspheres conjugated with anti-human IgE antibody were spotted in the labeled area on the top layer of the chip.

[0064] The biomolecular coating procedure for the chip substrate is as follows: First, 1.5 μL of streptavidin is sequentially spotted at the detection sites (T2, T4, T6, T8, T10, T12) on the chip substrate, incubated for 1 h, washed, and dried. Afterward, biotin-labeled food allergen proteins (egg, milk, soybean, peanut, wheat, cashew) are spotted at the same detection sites, and incubation continues for 1 h. After another wash, PE fluorescent microspheres conjugated with anti-human IgE antibody are spotted in the labeled area of ​​the chip substrate.

[0065] 3. Assembly of microfluidic chips

[0066] 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.

[0067] 4. Testing Procedure

[0068] First, move the flow control valve to the far right, disconnecting it from the microchannel. Add 20 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 surface tension and will not flow out spontaneously, while the liquid flowing to the left continues to flow. When it reaches the detection zone, the allergen sIgE antibody in the sample is captured by the corresponding allergen protein 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. Second, 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, the flow control valve is moved to the far left, embedding it within the microchannel. Buffer is added through the buffer injection well, entering the microchannel under capillary force. As it flows through the labeled area, it dissolves the fluorescent microspheres coupled with the anti-human IgE antibody. Upon reaching the detection area, the antibody binds to the specific IgE antibody already captured there. The remaining flow is collected in the flow control valve. Finally, a 488nm excitation source is used for excitation: ① fluorescence signals at 525nm are collected from each detection site in the top layer of the chip; ② the chip is excited again, and fluorescence signals at 575nm are collected from each detection site in the bottom layer.

[0069] 5. Test Results

[0070]

[0071] 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.

[0072] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0073] 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.

[0074] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "setting" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal communication between two components. Those skilled in the art will understand the specific meaning of the above terms in this invention based on the specific circumstances.

Claims

1. A microfluidic chip for increasing detection sites based on double color and double layer technology, comprising a substrate and a cover plate pressed on the substrate, the substrate and the cover plate enclosing a microchannel, the height of the microchannel being 20-50 μm, the left end of the microchannel being in communication with a buffer injection hole opened on the cover plate, the right end of the microchannel being provided with a liquid flow control valve, the liquid flow control valve controlling the opening and closing of the flow path of the liquid in the microchannel to the liquid flow control valve, the microchannel being sequentially provided with a labeling area and a detection area from left to right, characterized in that, The marking area includes a first marking area arranged on the lower surface of the cover sheet and a second marking area arranged on the upper surface of the base sheet, the detection area includes a plurality of detection sites arranged on the lower surface of the cover sheet and a plurality of detection sites arranged on the upper surface of the base sheet, and the detection sites arranged on the lower surface of the cover sheet and the upper surface of the base sheet do not coincide in the vertical plane, a sample injection hole is further arranged on the cover sheet, the sample injection hole is arranged between the detection area and the liquid flow control valve, a magnetic control intercept valve is arranged between the marking area and the detection area of the microchannel, the magnetic control intercept valve controls the opening and closing of the flow path between the marking area and the detection area in the microchannel through magnet adsorption, the first marking area is coated with a first marking antigen / antibody, the second marking area is coated with a second marking antigen / antibody, the first marking antigen / antibody and the second marking antigen / antibody are respectively two kinds of fluorescent microsphere markers with the same excitation spectrum but different emission spectrum, the plurality of detection sites are respectively coated with capture antigens / antibodies combined with different target analytes, a flow guide hole is arranged on the cover sheet, the bottom of the magnetic control intercept valve passes through the flow guide hole and contacts the upper surface of the base sheet, the top of the magnetic control intercept valve is made of ferrous material that can be adsorbed by a magnet, the bottom of the magnetic control intercept valve is separated from the flow guide hole after being adsorbed by the magnet, and the bottom of the magnetic control intercept valve is made of water-absorbing material. 2.The microfluidic chip based on double-color and double-layer technology for increasing detection sites according to claim 1, wherein, The first marking antigen / antibody is FITC fluorescent microsphere marking, and the second marking antigen / antibody is PE fluorescent microsphere marking. 3.The microfluidic chip for increasing detection sites based on double-color and double-layer technology according to claim 1, wherein, The lower surface of the cover sheet is provided with a groove along the length direction, and the cover sheet and the upper surface of the base sheet form a microchannel through the groove, and the width of the microchannel is 2-3 mm.

4. The microfluidic chip for increasing detection sites based on double-color and double-layer technology according to claim 1, wherein, The length of the detection area is 30 mm, and the distance between two adjacent detection sites in the same horizontal plane is greater than or equal to 5 mm.

5. The microfluidic chip for increasing detection sites based on double-color and double-layer technology according to claim 1, wherein, The distance between two adjacent detection sites in the same horizontal plane is the same. 6.The microfluidic chip for increasing detection sites based on double-color and double-layer technology according to claim 1, wherein, The number of detection sites arranged on the lower surface of the cover sheet is less than or equal to 6, and the number of detection sites arranged on the upper surface of the base sheet is less than or equal to 6.

7. The microfluidic chip for increasing detection sites based on double-color and double-layer technology 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 move away from the microchannel.

Images