Micro-fluidic chip capable of increasing detection sites based on double-color and double-layer technology and application of micro-fluidic chip

Abstract

The invention provides a micro-fluidic chip for increasing detection sites based on double-color and double-layer technology and application thereof, the micro-fluidic chip comprises a substrate and a cover plate pressed on the substrate, the substrate and the cover plate enclose to form a micro-channel, the micro-channel is provided with a marking area and a detection area, and the marking area and the detection area are arranged on the substrate. The marking area comprises a first marking area arranged on the lower surface of the cover plate and a second marking area arranged on the upper surface of the substrate, the detection area comprises a plurality of detection sites arranged on the lower surface of the cover plate and the upper surface of the substrate, the first marking area is coated with a first marking antigen / antibody, and the second marking area is coated with a second marking antigen / antibody. The first labeling area is coated with a first labeling antigen / antibody, the second labeling area is coated with a second labeling antigen / antibody, the first labeling antigen / antibody and the second labeling antigen / antibody are two fluorescent microsphere labels with the same excitation spectrum and different fluorescein emission spectrums, and the plurality of detection sites are respectively coated with capture antigens / antibodies combined with different target analytes. The number of synchronous detection indexes in the micro-channel is increased through layered design and combination of double-color marks.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of in vitro diagnosis and immunodetection, and particularly relates to a microfluidic chip based on double color and double layer technology for increasing detection sites and application thereof. BACKGROUND

[0002] Microfluidic technology refers to a technology for processing or manipulating microfluids by using microchannels of tens to hundreds of microns, which can integrate a series of complicated operations such as sample pretreatment, sample addition, reaction, separation and detection required for detection and analysis into a microfluidic chip of a few square centimeters, and can provide a closed environment required for in vitro diagnosis and detection, so as to realize low consumption, rapid and efficient analysis of samples. The biggest advantage is the flexible combination of various functional units, which not only realizes low reagent consumption and multi-index joint detection, but also has the advantages of easy integration and good compatibility with other technical equipment.

[0003] Immune microfluidic technology is a combination of immune analysis technology and microfluidic technology, which can make specific immune reactions proceed on a controlled microfluidic platform. Compared with traditional detection platforms, this technology integrates the antigen-antibody reaction process on the chip surface, and the micro-nano scale fluid control and integration not only improves the reaction speed of antigen and antibody, but also effectively shortens the reaction time, greatly simplifying the operation process of immune analysis.

[0004] Lateral flow immune microfluidic technology is a new instant detection platform that combines lateral flow immune analysis and microfluidic technology. The core is to replace the traditional nitrocellulose chromatography membrane with a micron-scale channel structure, and the antigen-antibody reaction is carried out in the micron-scale channel, which is formed by surrounding the substrate and the cover sheet. One end of the microchannel is in communication with the sample addition hole opened on the cover sheet, and the other end is in communication with the waste liquid collection pool. The microchannel is provided with a marking area, 1-6 detection points in the flow direction of the sample. The marking area in the substrate is pre-dried with labeled antibodies or antigens, and the multiple detection areas are indirectly coated with antigens or antibodies by the biotin-avidin system.

[0005] The demand for multi-index joint detection methods is increasingly urgent in clinical practice. For example, in the initial screening of allergens, the patient needs to be systematically screened for dozens of common allergens to determine the specific type of allergen that causes the patient's allergic reaction and provide a basis for personalized treatment. In addition, the detection of cytokines (such as IL-6, TNF-a, IFN-g, etc.) can reflect the activity of the immune system, which is of great significance for the diagnosis and prognosis evaluation of inflammatory reactions, tumor immunity, and various immune-related diseases. Furthermore, in the diagnosis of infectious diseases, the joint detection of inflammatory markers and various pathogens can help assess the degree of infection and identify the type of pathogen. Therefore, multi-index joint detection not only improves the efficiency of clinical diagnosis, but also provides doctors with more comprehensive information to develop more precise treatment plans.

[0006] However, methods such as chemiluminescence are often single-index detection followed by combined analysis. This approach not only has low efficiency, large sample consumption, but also has significant drawbacks such as cumulative operation errors and high costs. The lateral flow immunomicrofluidic technology, with its miniaturization and integration advantages, can achieve simultaneous detection of multiple target analytes in a single sample injection, effectively overcoming the limitations of traditional methods, significantly shortening the reporting time, and reducing sample and reagent consumption. It provides a strong technical platform to meet the clinical demand for efficient, accurate, and portable multi-index joint detection.

[0007] In existing lateral flow immunomicrofluidic chips, the length of the detection area is usually 30 mm, and the area of a single detection point is 1 mm 2 To avoid interference between the fluorescence signals of the detection points, the distance between two detection points must be ≥5 mm. Therefore, due to space limitations, this type of chip can at most achieve simultaneous detection of 6 indicators. To increase the number of detection indicators, the microchannel length must be extended, which leads to the following problems: ① increased manufacturing cost: extending the microchannel length requires the production of new chip molds, 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 length of the microchannel increases, the flow rate in the channel decreases non-linearly. In multi-index joint detection, the flow rate difference caused by the long channel leads to different reaction times at each detection point, affecting the consistency of the detection; ③ risk of flow stagnation: when the length of the microchannel exceeds the critical value of capillary action, capillary flow may be interrupted. SUMMARY

[0008] The application aims to provide a microfluidic chip based on double-color and double-layer technology for increasing detection sites and application thereof.

[0009] To achieve the above-mentioned purpose, the technical scheme of the application is as follows: a microfluidic chip based on double-color and double-layer technology for increasing detection sites, 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 communicated with a buffer injection hole opened on the cover plate, the right end of the microchannel is provided with a liquid flow control valve, the liquid flow control valve controls the opening and closing of the flow path of the liquid in the microchannel to the liquid flow control valve, the microchannel is sequentially provided with a marking area and a detection area from left to right, the marking area comprises a first marking area arranged on the lower surface of the cover plate and a second marking area arranged on the upper surface of the substrate, the detection area comprises a plurality of detection sites arranged on the lower surface of the cover plate and a plurality of detection sites arranged on the upper surface of the substrate, and the detection sites arranged 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 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 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 by 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 fluorescent microsphere markers of two kinds of fluorescent dyes with the same excitation spectrum but different emission spectra, and the plurality of detection sites are respectively coated with capture antigens / antibodies combined with different target analytes.

[0010] In an embodiment of the application, the first marking antigen / antibody is a FITC fluorescent microsphere marker, and the second marking antigen / antibody is a PE fluorescent microsphere marker.

[0011] In an embodiment of the application, the lower surface of the cover plate is provided with a groove along the length direction thereof, the cover plate and the upper surface of the substrate enclose the microchannel through the groove, and the width of the microchannel is 2-3 mm.

[0012] In an embodiment of the application, 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.

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

[0014] In one embodiment of the present application, 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 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 a ferrous material that can be attracted by a magnet, the magnetic control interception valve is attracted by magnetic force and the bottom is separated from the flow guide hole, and the bottom of the magnetic control interception valve is provided with a water-absorbing material.

[0016] In one embodiment of the present application, the liquid flow control valve is a movable water-absorbing material, which contacts or moves away from the microchannel after moving.

[0017] In another aspect, the present application also provides a use of the microfluidic chip in any one of the above technical solutions for multi-index detection, comprising at least the following steps: 1) The liquid flow control valve is moved to the rightmost position, 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 sides. The liquid flowing to the right is stopped at the end of the microchannel under the action of surface tension, while the liquid flowing to the left can continue to flow. When the sample to be tested in the sample to be tested contacts the capture antigen / antibody on the upper surface of the substrate and the lower surface of the cover sheet, respectively, an antigen-antibody complex is formed. The liquid flow is intercepted when it reaches the magnetic control interception valve, and then the liquid flow is collected into the magnetic control interception valve. After all the liquid flow in the channel is collected into the magnetic control interception valve, the interception valve is removed, and the microchannel returns to a smooth state. 2) The liquid flow control valve is moved to the leftmost position, so that it is embedded in the microchannel. The buffer solution is added to the buffer injection hole, which enters the microchannel under the action of capillary driving force, dissolves the labeled antigen / antibody in the labeled area on the upper surface of the substrate and the lower surface of the cover sheet, respectively, and binds the complex molecules in different detection sites on the top layer and the bottom layer of the chip, respectively. The remaining liquid flow is collected into the liquid flow control valve. 3) After the reaction is completed, the excitation light source is excited. First, the emission spectrum corresponding to the labeled fluorescein of the first labeled region fluorescent microsphere is used to collect the fluorescence signal of different detection sites on the lower surface of the cover sheet. Then, the emission spectrum corresponding to the labeled fluorescein of the second labeled region fluorescent microsphere is used to collect the fluorescence signal of different detection sites on the upper surface of the substrate.

[0018] The microfluidic chip based on the double-color and double-layer technology for increasing detection sites and the application thereof have the beneficial effects that: 1. Layered design, increasing the number of synchronous detection indexes in the microchannel: by taking the top layer of the chip and the bottom layer of the chip as two independent fluid reaction layers, 6 detection sites are respectively designed on the top layer and the bottom layer of the chip, and in the same physical space, the number of detection sites is doubled (12), the detection efficiency is improved, and the manufacturing cost is reduced.

[0019] 2. Double-color marking, eliminating signal interference: the top layer and the bottom layer of the chip are respectively marked with two kinds of fluorescent microsphere probes with the same excitation spectrum but different emission spectrum. The detection of the top layer of the chip is not interfered by the fluorescent signal of the adjacent bottom layer of the chip, and the detection of the bottom layer of the chip is not interfered by the fluorescent signal of the adjacent top layer of the chip. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a structure schematic diagram of the microfluidic chip (perspective) based on the double-color and double-layer technology for increasing detection sites according to the present application; Figure 2 is a structure schematic diagram of the cover sheet (perspective) according to the present application; Figure 3 is a structure schematic diagram of the substrate according to the present application; Figure 4 is a sectional view of the microfluidic chip based on the double-color and double-layer technology for increasing detection sites according to the present application; Figure 5 is a principle diagram of the microfluidic chip based on the double-color and double-layer technology for increasing detection sites according to the present application for dropping the sample to be detected; Figure 6 is a principle diagram of the microfluidic chip based on the double-color and double-layer technology for increasing detection sites according to the present application for dropping the buffer solution; Figure 7 is a detection site distribution diagram of the microfluidic chip for detecting the sIgE antibody of inhaled and ingested allergens according to the present application.

[0021] In the figure, 1 is a substrate, 2 is a cover sheet, 3 is a microchannel, 4 is a buffer solution injection hole, 5 is a liquid flow control valve, 6 is a sample injection hole, 7 is a magnetic control interception valve, 31 is a marking area, 31a is a first marking area, 31b is a second marking area, 32 is a detection area, and 32a is a detection site. DETAILED DESCRIPTION

[0022] 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.

[0023] Unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which the present application belongs. The experimental reagents used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods described are all conventional methods unless otherwise specified.

[0024] The present application relates to the field of immunoassay technology, and relates to a microfluidic chip based on two-color and double-layer technology to increase detection sites. The top layer of the chip and the bottom layer of the chip are used as two independent fluid reaction layers. Two fluorescent microsphere probes with the same excitation spectrum but different emission spectra (such as FITC fluorescent microspheres and PE fluorescent microspheres, both of which have an excitation wavelength of 488 nm, wherein the emission wavelength of FITC is 525 nm and the emission wavelength of PE is 575 nm) are respectively placed in the positions corresponding to the labeling area of the top layer of the chip and the bottom layer of the chip. Multiple detection sites are set up on the top layer of the chip and the bottom layer of the chip at the same time. The detection indexes can be increased in the microchannel of limited length, and the detection sites of the top layer and the bottom layer of the chip do not interfere with each other.

[0025] The present application will be further explained in conjunction with the examples and the accompanying drawings. It should be understood that the present application is not limited to the specific embodiments described.

[0026] As Figures 1-4As shown, the present application provides a microfluidic chip based on double color and double layer technology to increase detection sites, comprising a substrate and a cover sheet 2 pressed on the substrate 1, the substrate 1 and the cover sheet 2 form a microchannel 3, the height of the microchannel 3 is 20-50 μm, the left end of the microchannel 3 is communicated with the buffer injection hole 4 opened on the cover sheet 2, the right end of the microchannel 3 is provided with a liquid flow control valve 5, the liquid flow control valve 5 controls 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 is sequentially provided with a marking area 31 and a detection area 32 from left to right, the marking area 31 includes a first marking area 31a arranged on the lower surface of the cover sheet 2 and a second marking area 31b arranged on the upper surface of the substrate 1, the detection area 32 includes a plurality of detection sites 32a arranged on the lower surface of the cover sheet 2 and a plurality of detection sites 32a arranged on the upper surface of the substrate 1, and the detection sites 32a arranged on the lower surface of the cover sheet 2 and the upper surface of the substrate 1 do not coincide in the vertical plane, the cover sheet 2 is further provided with a sample injection hole 6, the sample injection hole 6 is arranged between the detection area 32 and the liquid flow control valve 5, the marking area 31 and the detection area 32 of the microchannel 3 are provided with a magnetic control intercept valve 7, the magnetic control intercept valve 7 controls the opening and closing of the flow path between the marking area 31 and the detection area 32 in the microchannel 3 by magnet adsorption, the first marking area 31a is coated with a first marking antigen / antibody, the second marking area 31b 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 and different emission spectrum fluorescein, and the plurality of detection sites 32a are respectively coated with capture antigens / antibodies combined with different target analytes.

[0027] In fluid mechanics, the Reynolds number (Re) is a key parameter to judge the flow state of liquid. When Re is lower than the critical value, the flow is laminar flow. In the microfluidic channel, Re < 100 can be regarded as laminar flow.

[0028] According to the calculation formula of Reynolds number: Re = (ρ is the fluid density, v is the average flow rate, Dh is the hydraulic diameter, μ is the dynamic viscosity of the fluid), for a rectangular microchannel, Dh = (w is the channel width, h is the channel height), since the width of the microchannel is much larger than the height, Dh can be simply written as Dh = 2h, so the height of the microchannel is the key parameter to determine the flow state of the liquid in the microchannel.

[0029] For common fluids (such as water, buffer), the channel height h is usually set to 10-100 μm at a typical flow rate (0.1-10 mm / s) to ensure Re<100. For other liquids with faster flow rate, the microchannel height is not properly designed, and the internal liquid flow will present irregular turbulent flow state, which will affect the reactions of different layers. Therefore, limiting the microchannel height to 20-50 μm can ensure that any liquid flow in the microchannel presents a smooth laminar flow state.

[0030] Therefore, in the lateral flow microchannel, when Re<100, the liquid flow presents a laminar flow state, and by virtue of this feature, the top layer of the chip and the bottom layer of the chip can be used as two independent fluid reaction layers.

[0031] Under the laminar flow-based microfluidic chip setting, 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, wherein the emission wavelength of FITC is 525 nm and the emission wavelength of PE is 575 nm) are placed at the positions of the corresponding labeling regions on the lower surface of the cover glass (the top layer of the chip) and the upper surface of the base plate (the bottom layer of the chip), respectively. Meanwhile, a plurality of 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 in the microchannel, and the detection sites on the top layer and the bottom layer are distributed in a staggered manner.

[0032] When starting the bidirectional immune microfluidic detection, first, the capture molecules (capture antigens or antibodies) located in the detection area bind to the molecules to be detected in the sample to be detected, respectively, to form antigen-antibody complexes; and then reverse lateral flow is started again to dissolve the labeled antigens or antibodies in the labeling regions of the top layer and the bottom layer of the chip, respectively, and bind to the complex molecules in different detection sites on the top layer and the bottom layer of the chip, respectively.

[0033] After the reaction is completed, the excitation light source is excited, and first, the emission spectrum corresponding to the labeling fluorescein on the lower surface of the cover glass (the top layer of the chip) is selected to collect the fluorescent signals of different detection sites on the top layer of the chip; and then the emission spectrum corresponding to the labeling fluorescein on the upper surface of the base plate (the bottom layer of the chip) is selected to collect the fluorescent signals of different detection sites on the bottom layer of the chip. Since the emission spectra of the top layer and the bottom layer of the chip are different, the detection of the top layer of the chip is not interfered by the fluorescent signals of the adjacent bottom layer of the chip; similarly, the detection of the bottom layer of the chip is also not interfered by the fluorescent signals of the adjacent top layer of the chip.

[0034] The first labeled antigen / antibody is FITC fluorescent microsphere labeling, and the second labeled antigen / antibody is PE fluorescent microsphere labeling.

[0035] 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.

[0036] 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.

[0037] The distance between two adjacent detection sites 32a on the same horizontal plane is the same.

[0038] 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.

[0039] 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.

[0040] The flow control valve 5 is made of a movable absorbent material, which moves to contact or move away from the microchannel.

[0041] 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: 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). Substrate: From left to right, these are the second marker area and the detection area. 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.

[0042] The specific workflow is as follows: A. Detection molecule capture stage: such as Figure 5As shown, the liquid flow control valve is set to the rightmost position, so that it is not connected to the microchannel. After the sample injection hole is added with the sample to be tested, 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 stays at the end of the microchannel under the 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 substance to be tested in the sample contacts the capture molecules on the top layer and the bottom layer of the chip to form an antigen-antibody complex. When the liquid flow reaches the magnetic control interception valve, it is intercepted. Since the water absorption provided by the magnetic control interception valve is greater than the capillary force in the microchannel, the liquid flow is collected in the magnetic control interception valve and does not continue to flow to the left. After the liquid flow in the microchannel is completely collected in the magnetic control interception valve, 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.

[0043] B. Labeling substance binding stage: as shown in Figure 6 The liquid flow control valve is set to the leftmost position, so that it is embedded in the microchannel. The buffer solution is added dropwise to the buffer injection hole, which enters the microchannel under the action of capillary driving force, dissolves the labeled antigen or antibody in the labeling area of the top layer and the bottom layer of the chip, respectively, and binds to the complex molecules in different detection sites of the top layer and the bottom layer of the chip, respectively. The remaining liquid flow is collected in the liquid flow control valve.

[0044] C. Excitation detection stage: after the reaction is completed, an excitation light source is used for excitation. First, the emission spectrum corresponding to the top layer of the chip is used to collect the fluorescence signals of different detection sites. Second, the emission spectrum corresponding to the bottom layer of the chip is used to collect the fluorescence signals of different detection sites. Since the emission spectrum of the top layer and the bottom layer of the chip is different, the detection of the top layer of the chip is not disturbed by the fluorescence signals of the adjacent bottom layer of the chip. Similarly, the detection of the bottom layer of the chip is not disturbed by the fluorescence signals of the adjacent top layer of the chip.

[0045] Example: Detection of inhaled and ingested allergens sIgE antibodies 1. Chip fabrication The substrate and the cover are both made of PMMA material. The microchannel structure, sample injection hole, buffer injection hole, liquid guide hole and other structures of the cover are designed by CAD software, then processed by CO2 laser etching machine, and finally the microchannel surface of the chip is treated for hydrophilicity.

[0046] 2. Biomolecule coating procedure Top layer of the chip biomolecule coating procedure: as shown in Figure 7As shown, first, 1.5 μL streptavidin was spotted on the detection sites (T1, T3, T5, T7, T9, T11) of the top layer of the chip in turn, incubated for 1 h and washed, dried and then biotin-labeled inhaled allergen proteins (house dust mites, dust mites, mugwort, pigweed, ragweed, mold) were spotted on the above detection sites respectively, and incubated for 1 h. After washing again, FITC fluorescent microsphere-coupled anti-human IgE antibody was spotted on the marker area of the top layer of the chip.

[0047] Chip bottom layer biomolecule coating procedure: First, 1.5 μL streptavidin was spotted on the detection sites (T2, T4, T6, T8, T10, T12) of the bottom layer of the chip in turn, incubated for 1 h and washed, dried and then biotin-labeled ingested allergen proteins (chicken egg, milk, soybean, peanut, wheat, cashew) were spotted on the above detection sites respectively, and incubated for 1 h. After washing again, PE fluorescent microsphere-coupled anti-human IgE antibody was spotted on the marker area of the bottom layer of the chip.

[0048] 3. Assembly of the microfluidic chip 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; finally, the top layer and the bottom layer of the chip were tightly attached and bonded.

[0049] 4. Detection procedure First, the liquid flow control valve was turned to the rightmost side so that it was not connected to the microchannel. 20 μL of the sample to be tested was added to the sample injection hole, and the sample flowed to the left and right sides. Since the microchannel was not connected to the liquid flow control valve, the liquid flowing to the right was stopped at the end of the microchannel under the action of the surface tension, and did not flow out spontaneously, while the liquid flowing to the left could continue to flow. When the sample to be tested reached the detection area, the allergen sIgE antibody in the sample was captured by the corresponding allergen protein in the detection area; the liquid flow was intercepted at the magnetic control intercept valve, and since the water absorption provided by the magnetic control intercept valve was greater than the capillary force in the microchannel, the liquid flow was collected into the magnetic control intercept valve, and did not continue to flow to the left. Second, after the liquid flow in the microchannel was completely collected into the magnetic control intercept valve, the intercept valve was removed, and the microchannel was restored to be unobstructed to ensure that the subsequent reagent flow and reaction were not disturbed. Third, the liquid flow control valve was turned to the leftmost side so that it was embedded in the microchannel, and buffer was added to the buffer injection hole, which entered the microchannel under the action of capillary driving force, dissolved the fluorescent microsphere-coupled anti-human IgE antibody when flowing through the marker area, and combined with the specific IgE antibody captured in the detection area when the liquid flow reached the detection area. The remaining liquid flow was collected into the liquid flow control valve. Finally, a 488 nm excitation light source was used for excitation: ① the fluorescence signals of each detection site in the top layer of the chip were collected at 525 nm; ② the fluorescence signals of each detection site in the bottom layer of the chip were collected at 575 nm.

[0050] 5. Detection result

[0051] The above technical solutions only reflect the preferred technical solutions of the present application, and some changes made by the 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.

[0052] 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.

[0053] In the description of the present application, it should be understood that the terms "center", "longitudinal", "transverse", "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer" and the like indicate the orientation or positional relationship shown in the drawings based on the orientation or positional relationship shown in the drawings, and are only for the purpose of facilitating the description of the present application and simplifying the description, and do not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application. In addition, the terms "first", "second" and the like are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined with "first", "second" and the like can explicitly or implicitly include one or more of the features. In the description of the present application, unless otherwise specified, the meaning of "a plurality of" is two or more.

[0054] In the description of the present application, it should be noted that unless otherwise explicitly specified and limited, the terms "mounting", "connecting", "connecting", "setting" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium; it can be the communication between the two elements inside. For those skilled in the art, the specific meaning of the above terms in the present application can be understood through specific circumstances.

Claims

1. A microfluidic chip for increasing detection sites based on dual-color and dual-layer technology, comprising a substrate and a cover plate pressed onto the substrate, wherein the substrate and the cover plate enclose a microchannel with a height of 20-50 μm, the left end of the microchannel communicating with a buffer injection hole on the cover plate, and a flow control valve disposed at the right end of the microchannel, the flow control valve controlling the opening and closing of the flow path of liquid in the microchannel to the flow control valve, wherein the microchannel is provided with a marking area and a detection area from left to right, characterized in that, The labeling area includes a first labeling area disposed on the lower surface of the cover plate and a second labeling area disposed on the upper surface of the substrate. The detection area includes multiple detection sites disposed on the lower surface of the cover plate and multiple detection sites disposed on the upper surface of the substrate. The detection sites disposed 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 is also provided with a sample injection hole, which is disposed between the detection area and the flow control valve. A magnetically controlled intercepting valve is disposed between the labeling area and the detection area of ​​the microchannel. The magnetically controlled intercepting valve uses a magnet to attract and control the opening and closing of the flow path between the labeling area and the detection area in the microchannel. The first labeling area is coated with a first labeled antigen / antibody, and the second labeling area 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.

2. The microfluidic chip based on dual-color and dual-layer technology to increase detection sites according to claim 1, characterized in that, The first labeled antigen / antibody is labeled with FITC fluorescent microspheres, and the second labeled antigen / antibody is labeled with PE fluorescent microspheres.

3. The microfluidic chip based on dual-color and dual-layer technology to increase detection sites according to claim 1, characterized in that, The cover plate has a groove along its length on its lower surface. The cover plate forms a microchannel by enclosing the upper surface of the substrate through the groove. The width of the microchannel is 2-3 mm.

4. The microfluidic chip based on dual-color and dual-layer technology to increase detection sites according to claim 1, characterized in that, The detection area is 30 mm long, and the distance between two adjacent detection sites on the same horizontal plane is greater than or equal to 5 mm.

5. The microfluidic chip based on dual-color and dual-layer technology to increase detection sites according to claim 1, characterized in that, The distance between two adjacent detection sites on the same horizontal plane is the same.

6. The microfluidic chip with increased detection sites based on dual-color and dual-layer technology according to claim 1, characterized in that, The cover plate has 6 or fewer detection sites on its lower surface and the substrate has 6 or fewer detection sites on its upper surface.

7. The microfluidic chip based on dual-color and dual-layer technology to increase detection sites according to claim 1, characterized in that, The cover plate is provided with a flow guide hole. The bottom of the magnetically controlled intercept valve passes through the flow guide hole and contacts the upper surface of the substrate. The top of the magnetically controlled intercept valve is provided with an iron material that can be attracted by a magnet. After the magnetically controlled intercept valve is attracted by magnetic force, the bottom of the magnetically controlled intercept valve is separated from the flow guide hole. The bottom of the magnetically controlled intercept valve is provided with a water-absorbing material.

8. The microfluidic chip with increased detection sites based on dual-color and dual-layer technology according to claim 1, characterized in that, The flow control valve is made of a movable absorbent material, which moves to contact or move away from the microchannel.

9. An application of a microfluidic chip that increases detection sites based on dual-color and dual-layer technology, characterized in that, Performing multi-index detection using the microfluidic chip according to any one of claims 1-8 includes at least the following steps: 1) Move the flow control valve to the far right so that it is not connected to the microchannel. After adding the sample to be tested into the sample injection hole, the sample flows to the left and right. The liquid flowing to the right stops at the end of the microchannel due to surface tension, while the liquid flowing to the left can continue to flow. When it reaches the detection area, the test substance in the sample comes into contact with the capture / antibody on the upper surface of the substrate and the lower surface of the cover plate, 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. 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. Under the action of capillary driving force, the buffer solution enters the microchannel and dissolves the labeled antigens / antibodies on the labeled areas of the upper surface of the substrate and the lower surface of the cover plate, respectively. The buffer solution binds 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. 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.

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