Microporous membrane permeation detection device based on immunolateral flow

Abstract

The present invention relates to a microporous membrane percolation detection device based on immune lateral flow, which includes an immune lateral flow channel and a microporous membrane with a pore size less than 1.2um, a solution outflow outlet is arranged at the end of the immune lateral flow channel, and the microporous membrane is attached to the solution outflow outlet; at least one labeling reagent is arranged in the immune lateral flow channel, and a capture reagent is coated on the microporous membrane. The present invention also discloses a microporous membrane percolation detection device, which includes an immune lateral flow channel and a microporous membrane with a substantially uniform pore size and a pore size greater than 20nm, a solution outflow outlet is arranged at the end of the immune lateral flow channel, and the microporous membrane is attached to the solution outflow outlet, and the microporous membrane is a hydrophilic microporous membrane; coated microspheres, a capture reagent fixed on the surface of the microspheres, and a labeling reagent are arranged in the immune lateral flow channel. Substances that are direct or indirect ligand relationships with each other are respectively arranged in the capture reagent and the labeling reagent.

Description

Technical Field

[0001] The invention belongs to the immune lateral flow detection technology in the field of biomedical diagnosis, and specifically relates to a microporous membrane percolation detection device based on immune lateral flow. Background Art

[0002] Immunolateral flow technology is a bioanalytical method developed at the end of the 20th century that combines immunoassay technology and chromatographic technology. This method has the characteristics of specificity, simple operation, and rapidity, and is widely used in important fields such as clinical diagnosis, environmental monitoring, and food safety. Traditional immunochromatographic technology uses colloidal gold as a marker and performs qualitative detection or semi-quantitative analysis of the target through strip color development. As a new type of immunoassay technology, fluorescent immunochromatography technology has the advantages of rapid on-site detection of traditional colloidal gold test strips and the high sensitivity of fluorescent technology.

[0003] Immunochromatography technology is based on antigen-antibody specific reactions. This technology uses a strip of fiber chromatography material with a hydrophilic porous membrane fixed with a test line (coated antibody or coated antigen, also recorded as T line) and a quality control line (anti-antibody, also recorded as C line) as the stationary phase, the test liquid as the mobile phase, and the labeled antibody or antigen fixed to the connection pad, and the analyte moves on the chromatography strip through capillary action. For macromolecular antigens with multiple antigenic determinants (proteins, viruses, pathogenic bacteria, etc.), a "sandwich" type double antibody sandwich immunochromatography method is usually used, that is, the analyte first binds to the fluorescent labeled antibody under the action of the mobile phase, and when it reaches the test line, it is dynamically captured and bound by the coated antibody to form a double antibody sandwich "sandwich" reaction complex.

[0004] The dot gold immunofiltration test is to drop purified antibodies in the center of the nitrocellulose membrane and adsorb them to the membrane. When the sample liquid dropped on the membrane is filtered through the membrane, the antigens in the sample are captured by the antibodies on the membrane, and the remaining irrelevant proteins are filtered out of the membrane. The colloidal gold marker added later also combines with the antigens bound to the membrane during the filtration. Because the colloidal gold itself is red, a positive reaction will show a red spot in the center of the membrane. Compared with the 5-20um pore size of the nitrocellulose membrane in the immune lateral chromatography, the membrane pore size in the filtration method is generally 0.45um, so the solution flow rate through the membrane is much slower, so immune reactions with weaker affinity can get suitable results in the filtration experiment. However, the dot gold immunofiltration experiment requires multiple solution addition operations. It would be better if it could be transformed into a one-step sample addition and simple operation.

[0005] The length of immunolateral chromatography on the solid phase membrane is generally more than 2cm, the width of the T line does not exceed 1mm, and the time for the liquid to move forward 1mm is only about 3 seconds. The protein to be tested must be captured and bound by the dynamic capture protein within 3 seconds. Therefore, the immune affinity of the coated protein is very high. Many proteins perform well in the static capture enzyme-linked immunosorbent assay (ELISA), but cannot be used in the application of immunolateral chromatography. Therefore, in many projects, the labeled antibody is pre-mixed with the sample solution, or the distance between the coating reagent and the labeled reagent is increased to allow for a more complete binding reaction. This can improve the detection sensitivity, but it adds additional operating steps. If the dynamic capture of proteins can be abandoned in the immunochromatography method and a mixed incubation method that is biased towards static incubation can be used for immunobinding capture, there will be better application effects.

[0006] Domestic patent document CN201310228708.8 discloses a quantitative detection device based on fiber membrane capture and separation and a detection method thereof: the coated microspheres of the labeled protein are placed in a deep-hole filter plate, and a static mixing and incubation reaction is carried out with the labeling reagent, and then the sample solution and the washing solution are drained from the filter membrane of the filter plate by centrifugal force, and small molecules or small-particle substances such as the labeling reagent flow out of the deep-hole filter plate with the solution, and the coated microspheres are partially intercepted when passing through the bottom filter membrane, and then the membrane surface is optically detected to obtain the relevant detection signal. The significance of this method is that the immune reaction reagent is statically incubated in a homogeneous environment, which further reduces the affinity requirements for the immune antibody antigen; at the same time, after the incubation reaction, the coated microspheres carrying the reaction complex in the solution are aggregated into the filter membrane, which plays a good role in concentrating the reaction substances. However, the relationship between the particle size of the coated microspheres and the pore size of the filter membrane in the patent document is not clearly defined, and the microspheres will penetrate into the filter membrane to a certain depth, which will cause some microspheres to pass through the filter membrane, thereby causing inaccurate detection results. At the same time, it will also lead to differences in the spectral signals reflected by the microspheres at different depths of the filter membrane.

[0007] Patent content

[0008] The purpose of the present invention is to provide a microporous membrane filtration detection device based on immune lateral flow, so as to achieve the capture and retention of the immune reaction complex of the immune chromatography test paper through the microporous membrane or microsphere capture and retention set in the immune lateral flow channel, and the signal of the immune reaction complex can be captured through the microporous membrane on the basis of one-step immune chromatography, thereby achieving a result with higher detection sensitivity.

[0009] To achieve the above object, the present invention adopts the following technical solution:

[0010] As the first aspect of the present invention, a microporous membrane filtration detection device based on immune lateral flow includes an immune lateral flow channel and a microporous membrane with a pore size less than 1.2um. The solution in the immune lateral flow channel directly flows into a solution outlet arranged at its end, and the microporous membrane is attached to the solution outlet.

[0011] According to the present invention, at least one labeling reagent is arranged in the immune lateral flow channel, and a capture reagent is coated on the microporous membrane; the capture reagent and the labeling reagent are respectively provided with substances that are direct or indirect ligands to each other.

[0012] According to the present invention, the immunolateral flow channel comprises a hydrophilic porous membrane capable of directionally transmitting an aqueous solution, wherein the hydrophilic porous membrane is selected from one or more of a nitrocellulose membrane, a glass fiber membrane, a chemical fiber membrane and a filter paper;

[0013] The immunolateral flow channel also includes a void channel constructed by sandwiching the hydrophilic porous membrane between the hydrophilic surfaces of two hydrophilic membranes, and the solution flows from one end of the void channel to the other end.

[0014] According to the present invention, the upper hydrophilic film of the two hydrophilic films is transparent, and the gap channel constructed between the upper hydrophilic film and the microporous membrane guides the solution to flow into the microporous membrane.

[0015] According to the present invention, the labeling reagent includes color particles or fluorescent substances that are directly visible to the naked eye; the color particles are selected from one or more of nano-colloidal gold, latex microspheres, carbon black particles, other nanoparticles with color signals, etc.; the fluorescent substance is selected from one or more of fluorescent molecules and their microspheres, quantum dot particles and their microspheres, up-conversion luminescent particles and their microspheres, time-resolved fluorescent molecules and their microspheres, fluorescent proteins, etc.

[0016] According to the present invention, the microporous membrane filtration detection device based on immune lateral flow also includes a detection device that can irradiate the reaction complex accumulated on the microporous membrane after the immune lateral flow with light of a specific wavelength and receive at least one wavelength spectral signal reflected after being excited.

[0017] As the second aspect of the present invention, a microporous membrane filtration detection device based on immune lateral flow includes an immune lateral flow channel and a microporous membrane with basically uniform pore size in the range of 0.02-1.2um. The solution in the immune lateral flow channel flows to a solution outlet arranged at the end thereof, and the microporous membrane is attached to the solution outlet. The microporous membrane is hydrophilic and the solution can pass through the filtration.

[0018] According to the present invention, at least one coated microsphere with a substantially uniform diameter larger than the pore size of a microporous membrane, a capture reagent fixed on the surface of the microsphere, and a labeling reagent with a diameter smaller than the pore size of the microporous membrane are arranged in the immune lateral flow channel; the capture reagent and the labeling reagent are respectively provided with substances that are direct or indirect ligand relationships with each other.

[0019] According to the present invention, the immunolateral flow channel comprises a hydrophilic porous membrane capable of directionally transmitting an aqueous solution, wherein the hydrophilic porous membrane is selected from one or more of a nitrocellulose membrane, a glass fiber membrane, a chemical fiber membrane and a filter paper;

[0020] The immunolateral flow channel also includes a void channel constructed by sandwiching the hydrophilic porous membrane between the hydrophilic surfaces of two hydrophilic membranes, and the solution flows from one end of the void channel to the other end.

[0021] According to the present invention, two hydrophilic membranes are arranged at the end of the immune lateral flow channel, the hydrophilic surfaces of the two hydrophilic membranes clamp the upper and lower surfaces of the hydrophilic porous membrane, the lower surface of the hydrophilic membrane at the bottom of the two hydrophilic membranes is attached to the microporous membrane, and a notch is provided in the middle of the hydrophilic membrane attached to the upper surface of the microporous membrane as a solution outflow outlet;

[0022] Alternatively, the hydrophilic surfaces of the two hydrophilic membranes clamp the left and right sides of the hydrophilic porous membrane, and a notch is provided in the middle of the bottom surface of the gap channel formed by the two hydrophilic membranes as a solution outflow outlet.

[0023] According to the present invention, the notch shapes provided on the hydrophilic membrane at least include a gap shape with a maximum gap not exceeding 1.5 mm or an angle not exceeding 95 degrees.

[0024] According to the present invention, a water-absorbing material is attached to the lower surface of the microporous membrane, and the solution flowing into the gap of the hydrophilic membrane passes through the microporous membrane and is absorbed and transferred by the water-absorbing material.

[0025] According to the present invention, the labeling reagent includes color particles or fluorescent substances that are directly visible to the naked eye; the color particles are selected from one or more of nano-colloidal gold, latex microspheres, carbon black particles, other nanoparticles with color signals, etc.; the fluorescent substance is selected from one or more of fluorescent molecules and their microspheres, quantum dot particles and their microspheres, up-conversion luminescent particles and their microspheres, time-resolved fluorescent molecules and their microspheres, fluorescent proteins, etc.

[0026] Furthermore, the microporous membrane filtration detection device based on immunolateral flow also includes a detection device that can irradiate the reaction complex trapped and accumulated in the microporous membrane after immunolateral flow with light of a specific wavelength and receive at least one wavelength spectral signal reflected after being excited.

[0027] According to the present invention, when the coated microspheres and the microspheres formed by the reaction complexes with the labeling reagents are accumulated on the surface of the microporous membrane, the free labeling reagents can be carried by the solution through the pores between the accumulated microspheres and then flow out from the micropores of the microporous membrane again without forming any interception residues.

[0028] The microporous membrane percolation detection device based on immune lateral flow of the present invention has the following effects: it can capture the microsphere reaction complex of the immune chromatographic test paper through the microporous membrane set in the lateral flow channel or the microsphere capture, interception and aggregation, and can capture and intercept the signal of the immune reaction complex on the basis of one-step immune chromatography, thereby achieving a result with higher detection sensitivity. The details are as follows:

[0029] 1. Improved reaction sensitivity: When using microporous membrane as coating carrier, the flow rate of microporous membrane solution with pore size less than 1.2um will slow down, thus reducing the dependence of dynamic reaction on the affinity of ligands such as antibodies. At the same time, the protein binding capacity of microporous membranes with smaller pore size, such as nitrocellulose membrane, will increase, and the increased protein density is also conducive to the capture ability of coated protein on microporous membrane; when using microspheres as coating carrier, it fundamentally changes the dynamic capture mode of immune response complexes, reducing the dependence on ligand affinity in immune response. The coating reagent and the labeling reagent are fully mixed and react freely in the flow of solution with the sample to be tested. The reaction is no longer captured by the coating protein of the microporous membrane on the ligand of dynamic chromatography, but the reaction complex is intercepted and aggregated by the microporous membrane, thereby improving the sensitivity of the reaction;

[0030] 2. The reaction complexes retained by the microporous membrane are concentrated in the same position, providing an opportunity for single-point multiplex immunofluorescence detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a schematic structural diagram of the microporous membrane filtration detection device based on immune lateral flow of the present invention.

[0032] Figure 2 Schematic diagram of the combination of the sample to be tested, the microspheres with the capture reagent fixed thereon and the labeling reagent.

[0033] Figure 3 Another schematic diagram of the combination of the sample to be tested, the microspheres immobilized with the capture reagent and the labeling reagent.

[0034] Figure 4 Another structural schematic diagram of a microporous membrane filtration detection device based on immune lateral flow.

[0035] Figure 5 It is a schematic diagram of a partial top view of a microporous membrane filtration detection device based on immune lateral flow.

[0036] Figure 6It is a schematic diagram of the structure of the two hydrophilic membranes of another microporous membrane filtration detection device.

[0037] Figure 7 It is a schematic structural diagram of the solution outflow outlet of the present invention.

[0038] Figure 8 Another schematic diagram of the structure of the solution outflow outlet of the present invention. DETAILED DESCRIPTION

[0039] In order to make the above-mentioned purposes, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the drawings of the specification. Obviously, the described embodiments are part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary persons in the art without creative work should fall within the scope of protection of the present invention.

[0040] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0041] Secondly, the present invention is described in detail with reference to schematic diagrams. When describing the embodiments of the present invention in detail, for the sake of convenience, the cross-sectional diagrams showing the device structure will not be partially enlarged according to the general scale, and the schematic diagrams are only examples, which should not limit the scope of protection of the present invention. In addition, in actual production, the three-dimensional dimensions of length, width and depth should be included.

[0042] At the same time, in the description of the present invention, it should be noted that the orientations or positional relationships indicated by the terms "upper, lower, inside and outside" are based on the orientations or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0043] like Figure 1 As shown, a microporous membrane filtration detection device based on immune lateral flow of the present invention comprises an immune lateral flow channel and a microporous membrane 3 with a pore size less than 1.2 um. The solution in the immune lateral flow channel directly flows into a solution outflow outlet 9 arranged at the end thereof. The solution outflow outlet 9 is attached to the microporous membrane 3 for the solution to pass through the filtration, or the solution that reaches the microporous membrane by lateral flow will change direction and flow downward through the microporous membrane; at least one labeling reagent is arranged in the immune lateral flow channel, and a capture reagent is coated on the microporous membrane 3; substances that are direct or indirect ligands to each other are respectively arranged in the capture reagent and the labeling reagent.

[0044] In a competitive immune reaction, the capture reagent can be a competitive antigen coated on a microporous membrane, such as a small molecule antigen coupled to a carrier protein, and the labeling reagent is an antibody to the substance to be tested labeled with fluorescence or other spectral signals; when the free antigen in the solution reacts with all the labeling reagents in the solution, the capture reagent will no longer have an immune reaction complex; when the free antigen in the solution reacts with part of the labeling reagent in the solution, the capture reagent on the microporous membrane will produce a partial immune reaction complex, so a part of the immune reaction complex will be captured on the microporous membrane.

[0045] In the immunosandwich reaction, the capture reagent can be antibody A coated on the microporous membrane, and the labeling reagent can be antibody B labeled with fluorescence or other spectral signals; when the antigen in the solution flows in the lateral flow solution, it first reacts with the labeling reagent and antibody B dissolved in the solution at the same time, and when the solution percolates and flows on the microporous membrane, it is captured by antibody A on the microporous membrane and retained on the microporous membrane. The immunolateral flow channel includes a hydrophilic porous membrane 1 that can directional transmit an aqueous solution, and the hydrophilic porous membrane is selected from one or more of nitrocellulose membrane, glass fiber membrane, chemical fiber membrane and filter paper.

[0046] The immunolateral flow channel also includes a void channel constructed by the hydrophilic surfaces of the upper and lower hydrophilic membranes 4 clamping the hydrophilic porous membrane 1, and the surface tension of the solution on the hydrophilic membrane surface is used as the driving force for liquid flow, and the solution flows from one end of the void channel to the other end.

[0047] The upper hydrophilic film of the two hydrophilic films 4 is transparent, and the gap channel constructed between it and the microporous membrane 3 guides the solution to flow into the microporous membrane 3.

[0048] The lower surface of the microporous membrane 3 is adhered with a water-absorbing material 2, such as filter paper. The solution flowing into the hydrophilic membrane 4 from the gap passes through the microporous membrane 3 and is absorbed and transferred by the water-absorbing material 2, such as the filter paper.

[0049] The labeling reagent includes color particles or fluorescent substances that are directly visible to the naked eye; the color particles are selected from one or more of nano-colloidal gold, latex microspheres, carbon black particles, other nanoparticles with color signals, etc.; the fluorescent substance is selected from one or more of fluorescent molecules and their microspheres, quantum dot particles and their microspheres, up-conversion luminescent particles and their microspheres, time-resolved fluorescent molecules and their microspheres, fluorescent proteins, etc.

[0050] The microporous membrane percolation detection device based on immune lateral flow also includes a detection device that can irradiate the immune reaction complex captured and accumulated on the surface of the microporous membrane 3 after the immune lateral flow with light of a specific wavelength and receive at least one reflected wavelength spectrum signal after being excited. When the immune reaction complex accumulated on the surface of the microporous membrane 3 is irradiated with light of a specific wavelength, at least one fluorescent marker substance in the reaction complex is excited to emit a fluorescent signal of different wavelengths, which is received by the fluorescence spectrometer and then analyzed by a microprocessor to detect different microsphere reaction complexes, and the concentrations of different objects to be detected are calculated.

[0051] like Figure 1 As shown, the front end of the hydrophilic porous membrane 1 is also provided with a pretreatment microporous membrane 8 having a pore size smaller than that of the microporous membrane 3; the sample solution flows from the sample absorption pad 6 through the pretreatment microporous membrane 8 and then flows to the hydrophilic porous membrane 1; the hydrophilic porous membrane fixed with the labeling reagent is arranged at the rear end of the pretreatment microporous membrane 8, and is dissolved by the solution of the pretreatment microporous membrane 8 and then flows through the hydrophilic porous membrane.

[0052] like Figure 4 and Figure 5 As shown, another microporous membrane percolation detection device based on immune lateral flow of the present invention comprises an immune lateral flow channel and a microporous membrane 3 with a substantially uniform pore size and a pore size range of 0.02-1.2um. The microporous membrane 3 is hydrophilic, and it allows the solution to pass through the percolation, or the solution that flows laterally to the microporous membrane will change direction and flow downward through the microporous membrane; the solution in the immune lateral flow channel flows to the solution outflow outlet 9 set at its end, and the microporous membrane 3 is attached to the solution outflow outlet 9; at least one coated microsphere with a substantially uniform diameter and larger than the pore size of the microporous membrane 3, a capture reagent fixed on the surface of the microsphere, and a labeling reagent with a diameter smaller than the pore size of the microporous membrane are set in the immune lateral flow channel; the capture reagent and the labeling reagent are respectively provided with substances that are direct or indirect ligand relationships with each other. The capture reagent, the labeling reagent, and the sample to be tested undergo a binding reaction in the lateral flow of the solution to form a reaction complex on the surface of the microsphere.

[0053] The labeled reagent can be suspended in a solution and added to the lateral flow channel; the labeled reagent can also be coated and solidified on a porous membrane in the flow channel or coated and solidified on a surface of the lateral flow channel, and then dissolved by the solution and flow forward with the solution. The coated microspheres can be suspended in a solution and added to the lateral flow channel; the coated microspheres can also be coated and solidified on a porous membrane in the flow channel or coated and solidified on a surface of the lateral flow channel, and then dissolved by the solution and flow forward with the solution.

[0054] In the competitive immune reaction, the capture reagent can be a competitive antigen coated on the microspheres, such as a small molecule antigen coupled to a carrier protein, and the labeling reagent is an antibody to the test substance labeled with fluorescence or other spectral signals; when the free antigen in the solution reacts with all the labeling reagents in the solution, the capture reagent will no longer have an immune reaction complex, so the free coated microspheres will be retained on the surface of the microporous membrane; when the free antigen in the solution reacts with part of the labeling reagent in the solution, the capture reagent will produce part of the immune reaction complex, so the free coated microspheres and part of the microspheres with immune reaction complexes will be retained on the surface of the microporous membrane.

[0055] The clenbuterol competitive antigen protein was coated on PS microspheres with a diameter greater than 800nm ​​and solidified on the sample pad; clenbuterol monoclonal antibody was labeled with fluorescent particles with a diameter less than 300nm and solidified in the reagent binding pad; when the sample solution was added to the sample pad, the sample solution first reacted with the coated microspheres coated with the clenbuterol competitive antigen protein, and then the solution flowed laterally through the sample pretreatment membrane with a uniform pore size of 450nm, gradually dissolving and mixing the fluorescent particles labeled with clenbuterol monoclonal antibody on the reagent binding pad. After the confluence, the mixed reaction passes through a certain length of chromatographic membrane. If the free clenbuterol in the solution reacts with all the labeled reagents in the solution, the capture reagent coated with the microspheres will no longer have immune reaction complexes, so the free PS microspheres will be retained on the surface of the microporous membrane; when the free clenbuterol in the solution reacts with part of the labeled reagents in the solution, the capture reagent will produce part of the immune reaction complexes, so the free coated microspheres and part of the PS microspheres with clenbuterol immune reaction complexes will be retained on the surface of the microporous membrane.

[0056] In the immune sandwich reaction, the capture reagent can be antibody A coated on PS microspheres, and the labeling reagent is antibody B labeled with fluorescence or other spectral signals; when the antigen in the solution is captured by antibody A and antibody B at the same time in the solution, the free PS microspheres and PS microspheres with immune reaction complexes will be retained on the surface of the microporous membrane. HCG monoclonal antibody A is coated on PS microspheres with a diameter greater than 800nm ​​and solidified on the sample pad; HCG monoclonal antibody B is labeled with fluorescent particles with a diameter less than 300nm and solidified in the reagent binding pad; when the sample solution is added to the sample pad, the sample solution first dissolves and mixes the PS microspheres coated with HCG monoclonal antibody A, and then the solution flows laterally through the sample pretreatment membrane with a uniform pore size of 450nm, gradually dissolving and mixing the fluorescent particles labeled with HCG monoclonal antibody B on the reagent binding pad and flowing forward, after the mixing reaction of the chromatographic membrane, when the HCG protein in the solution is captured by antibody A and antibody B in the solution to form an immune response complex, the free coated microspheres and the microspheres with the immune response complex will be retained on the surface of the microporous membrane.

[0057] like Figure 1 , Figure 4 and Figure 5 As shown, the immune lateral flow channel includes a hydrophilic porous membrane 1 that can directional transmit an aqueous solution, and the hydrophilic porous membrane 1 is selected from one or more of a nitrocellulose membrane, a glass fiber membrane, a chemical fiber membrane, and a filter paper; the immune lateral flow channel also includes a gap channel constructed by clamping the hydrophilic porous membrane by the hydrophilic surfaces of the upper and lower hydrophilic membranes 4, and the surface tension of the solution generated by the hydrophilic surface of the hydrophilic membrane 4 is used as the power of liquid flow, and the solution flows from one end of the gap channel to the other end. The sample absorption pad glass fiber membrane can be used as a sample absorption pad (to be tested) and a reagent binding pad (marker, standard, microspheres connected to a capture reagent, etc.), the chemical fiber membrane acts as a hydrophilic membrane to guide the waterway, and the filter paper can be used as a water-absorbing material.

[0058] The lower surface of the hydrophilic membrane 4 attached to the microporous membrane 3 in the immune lateral flow channel is coated with glue or adhesive, and the microspheres in the solution will not flow away from the gap between the hydrophilic membrane and the microporous membrane, but can only flow out from the microporous membrane 3 attached to the gap of the hydrophilic membrane as the solution outflow outlet 9. Figure 7 As shown, the notch may be polygonal, circular or rectangular, and the notch may have at least one gap shape 91 (eg, Figure 7 and Figure 8 As shown) or an angle not exceeding 95 degrees, it is more conducive for the solution flowing to the edge of the gap to flow into the gap along the side and contact the microporous membrane with good hydrophilicity attached to the bottom of the side of the hydrophilic membrane; when the gap becomes a rectangle, its width can be set to 0.2-2.5mm, and the solution on the upper surface of its two sides will pull the solution from the side to the front across the gap due to surface tension. At this time, under the action of gravity, the solution that has crossed the side will sink and contact the hydrophilic porous membrane at the bottom, and then under the action of the hydrophilic pulling force of the hydrophilic porous membrane, the liquid will enter the gap and pass through the microporous membrane 3.

[0059] When in use, the hydrophilic surface of the hydrophilic membrane 4 can transfer the solution to the edge of the gap, but the side of the gap of the hydrophilic membrane 4 is difficult to process to achieve the same surface hydrophilicity, so the solution is not easy to flow into the gap along the side and contact the microporous membrane with good hydrophilicity attached to the bottom of the side of the hydrophilic membrane. When the critical condition caused by the gap or angle of the gap is reached, that is, the gap shape with a maximum gap of no more than 1.5mm or the angle of no more than 95 degrees is set, the solution on the upper surface of the two adjacent sides will pull the solution forward from the side to cross the gap due to surface tension. At this time, under the action of gravity, the solution that has crossed the side will sink and contact the hydrophilic microporous membrane at the bottom, and then under the action of the hydrophilic pulling force of the microporous membrane, the liquid will enter the gap and pass through the microporous membrane 3. When the lateral flow channel is formed by two layers of hydrophilic membranes, at least the hydrophilic membrane opposite to the hydrophilic membrane with the gap is transparent, and the surface of the microporous membrane at the gap of the opposite hydrophilic membrane can be detected by fluorescence or other spectra.

[0060] like Figure 1 As shown in FIG4 , two hydrophilic membranes are disposed at the end of the immune lateral flow channel, and the hydrophilic surfaces of the two hydrophilic membranes clamp the upper and lower surfaces of the hydrophilic porous membrane. The lower surface of the hydrophilic membrane at the bottom of the two hydrophilic membranes is attached to the microporous membrane, and a notch is provided in the middle of the hydrophilic membrane attached to the upper surface of the microporous membrane as a solution outflow outlet 9. Figure 6 As shown, the hydrophilic surfaces of the two hydrophilic membranes can also clamp the left and right sides of the hydrophilic porous membrane, and a notch is provided in the middle of the bottom surface of the gap channel formed by the two hydrophilic membranes as a solution outflow outlet 9. In order to construct a gap channel, the two hydrophilic membranes can clamp the hydrophilic porous membrane 1 to form a gap channel, and a double-sided tape or other blocking materials can be attached to the end of the channel to fix and block the gap between the two hydrophilic membranes, so that the solution in the gap channel no longer flows out.

[0061] The lower surface of the microporous membrane 3 is attached with a water-absorbing material 2, such as filter paper, etc. The solution flowing into the hydrophilic membrane 4 from the gap passes through the microporous membrane 3 and is absorbed and transferred by the water-absorbing material 2, such as the filter paper. There are also microporous membranes formed directly on the surface of the filter paper in the market, and this commercial product can be directly used to attach the hydrophilic membrane with glue on the surface to the surface of the microporous membrane, which can better ensure the water absorption effect.

[0062] like Figure 4 As shown, the front end of the hydrophilic porous membrane 1 is also provided with a pretreatment microporous membrane 8 having a pore size smaller than that of the microporous membrane; the sample solution flows from the sample absorption pad 6 through the pretreatment microporous membrane 8 and then flows to the hydrophilic porous membrane; the coated microspheres fixed with the capture reagent are arranged at the rear end of the pretreatment microporous membrane 8, and are dissolved by the solution of the pretreatment microporous membrane 8 and then flow through the hydrophilic porous membrane.

[0063] The labeling reagent includes color particles or fluorescent substances that are directly visible to the naked eye; the color particles are selected from one or more of nano-colloidal gold, latex microspheres, carbon black particles, and other nanoparticles with color signals; the fluorescent substance is selected from one or more of fluorescent molecules and their microspheres, quantum dot particles and their microspheres, up-conversion luminescent particles and their microspheres, time-resolved fluorescent molecules and their microspheres, and fluorescent proteins.

[0064] The microporous membrane percolation detection device based on immunolateral flow also includes a detection device capable of irradiating the reaction complex trapped and accumulated on the microporous membrane after the immunolateral flow with light of a specific wavelength and receiving at least one reflected wavelength spectrum signal after being excited. When the reaction complex accumulated on the surface of the microporous membrane is irradiated with light of a specific wavelength, at least two fluorescent markers in the reaction complex are excited to emit fluorescent signals of different wavelengths, which are received by the fluorescence spectrometer and analyzed by a microprocessor to detect different microsphere reaction complexes, and the concentrations of different objects to be detected are calculated.

[0065] When the coated microspheres and the microspheres formed by the reaction complexes with the labeling reagents are accumulated on the surface of the microporous membrane, the free labeling reagents can be carried by the solution through the pores between the accumulated microspheres and then flow out of the micropores of the filter membrane again without forming any blocking residues.

[0066] like Figure 4 As shown, a microporous membrane-entrapped microsphere detection device based on immune lateral flow of the present invention is attached. A nitrocellulose membrane (i.e., a porous hydrophilic membrane 1) is attached to a PVC bottom plate coated with a self-adhesive sticker, and a reagent binding pad 7, a pretreatment microporous membrane 8, and a sample absorption pad 6 are attached to the lower edge of the front end in sequence, wherein the sample absorption pad is 16 mm long and overlaps with the pretreatment microporous membrane 8 by 2 mm, and the pretreatment microporous membrane 8 with a length of 12 mm overlaps with the reagent binding pad 7 by 2 mm, completely covering the overlapped portion of the sample absorption pad 6 and the reagent binding pad 7; the reagent binding pad 7 with a length of 8 mm overlaps with the nitrocellulose membrane with a length of 20 mm by 1 mm, and the nitrocellulose membrane is sandwiched by the upper and lower hydrophilic membranes 4 by 1-10 mm, and the length of the hydrophilic membrane gap channel formed is 5-30 mm, and the position of the notch at the bottom of the hydrophilic membrane 4 is as shown in FIG. Figure 4 As shown in FIG. 1 (the notch 9 is provided on the lower hydrophilic membrane), the width of the notch is 0.2-1.5 mm, the intercepting microporous filter membrane is closely attached to the notch of the hydrophilic membrane, and the width of the microporous filter membrane is 2-8 mm. When the two are assembled, Figure 4 As shown, at this time, the sample solution flows from the immune lateral flow channel to the microporous membrane 3, and all the solution can only flow through the microporous membrane 3 from the gap of the hydrophilic membrane 4.

[0067] In this embodiment, there are three ways for the capture reagent, labeling reagent, and sample to be tested to undergo a binding reaction in the solution:

[0068] (1) The sample to be tested competes with the labeled reagent to bind to the microspheres immobilized with the capture reagent in the reaction zone of the solution, forming a reaction complex on the surface of the microspheres. After the solution is fully reacted, when the reaction solution flows through the microporous membrane, the microspheres immobilized with the capture reagent and the reaction complex are retained on the surface of the microporous membrane;

[0069] (2) Figure 2 As shown, the sample 15 to be tested competes with the microsphere 11 fixed with the capture reagent 12 for binding to the labeling reagent 14, and a reaction complex 13 is formed on the surface of the microsphere 11. After the solution is fully reacted, when the reaction solution flows through the microporous membrane, the microsphere 11 fixed with the capture reagent 12 and the reaction complex 13 are retained on the surface of the microporous membrane;

[0070] (3) Figure 3 As shown, the sample 15 to be tested, the microsphere 11 with the capture reagent 12 fixed thereon, and the labeling reagent 14 combine to form a reaction complex 13. After the solution is fully reacted, when the reaction solution flows through the microporous membrane, the microsphere 11 with the capture reagent 12 fixed thereon and the reaction complex 13 are retained on the surface of the microporous membrane.

[0071] Example 1: Microporous membrane detection device based on immunolateral flow for detecting HCG protein in urine

[0072] A microporous membrane percolation detection device based on immune lateral flow, comprising an immune lateral flow channel and a microporous membrane with a pore size of 0.45um. The immune lateral flow channel is mainly composed of a sample absorption pad, a reagent binding pad, a hydrophilic porous membrane, and a microporous membrane. The sample absorption pad, the reagent binding pad, the hydrophilic porous membrane, and the microporous membrane are arranged in sequence from front to back. The hydrophilic porous membrane is formed by clamping the hydrophilic surfaces of two layers of hydrophilic membranes facing each other, and maintaining a gap of 0.15mm extending forward by about 5mm. The solution of the hydrophilic porous membrane is drained out by the hydrophilic membranes clamping the hydrophilic porous membrane above and below, and the solution directly flows into a solution outflow port 9 arranged at the end of the bottom hydrophilic membrane, and a 0.45um microporous membrane 3 is attached to the solution outflow port 9.

[0073] A colloidal gold particle labeled with HCG monoclonal antibody A is added to the reagent binding pad of the immune lateral flow channel, and the microporous membrane is coated with HCG monoclonal antibody B; when the solution contains HCG protein and flows in the immune lateral flow channel, the HCG protein will be reacted and bound by HCG monoclonal antibody A, and then when passing through the microporous membrane 3, the reaction complex will be captured by HCG monoclonal antibody B again, forming two monoclonal antibody sandwiched HCG protein reaction complexes on the membrane surface of the microporous membrane. These reaction complexes can be used to judge and analyze the concentration of HCG in the solution through the user's naked eyes or the matching colloidal gold quantitative analysis instrument.

[0074] Example 2: Microporous membrane microsphere detection device based on immune lateral flow is used to detect HCG protein in urine

[0075] A microporous membrane percolation and microsphere retention detection device based on immune lateral flow, comprising an immune lateral flow channel and a microporous membrane with a pore size of 0.22um. The immune lateral flow channel is mainly composed of a sample absorption pad, a reagent binding pad, a hydrophilic porous membrane and a microporous membrane. The sample absorption pad, the reagent binding pad, the hydrophilic porous membrane and the microporous membrane are arranged in sequence from front to back. The hydrophilic porous membrane is formed by clamping the hydrophilic surfaces of two layers of hydrophilic membranes facing each other, and maintaining a gap of 0.15mm extending forward by about 5mm. The solution of the hydrophilic porous membrane is drained out by the hydrophilic membranes clamping the hydrophilic porous membrane above and below, and the solution directly flows into a solution outflow outlet arranged at the end of the bottom hydrophilic membrane, and a 0.22um microporous membrane is attached to the solution outflow outlet.

[0076] The sample absorption pad in the immune lateral flow channel is solidified with colloidal gold particles with a particle size of about 40nm marked with HCG monoclonal antibody A, and the reagent binding pad is solidified with coated microspheres with a diameter of 1.5um and fixedly coated with HCG monoclonal antibody A on the surface. When the urine sample solution contains HCG protein and flows in the immune lateral flow channel, the HCG protein will react and combine with the microspheres and nano-gold particles corresponding to HCG monoclonal antibody A and monoclonal antibody B during the solution chromatography process to form a reaction complex of two monoclonal antibody sandwiched HCG protein. Subsequently, when passing through the 0.22um microporous membrane, the free colloidal gold particles with a particle size of about 40nm marked with HCG monoclonal antibody A will pass through the microporous membrane and flow away, and the coated microspheres of the reaction complex and the free coated microspheres will be retained on the surface of the microporous membrane. These reaction complexes can be used to judge and analyze the concentration of HCG in the solution through the user's naked eyes or the matching colloidal gold quantitative analysis instrument.

[0077] Example 3: Microporous membrane microsphere detection device based on immunolateral flow is used to detect FOB protein in fecal samples

[0078] A microporous membrane percolation and microsphere retention detection device based on immune lateral flow, comprising an immune lateral flow channel and a microporous membrane with a pore size of 0.22um. The immune lateral flow channel is mainly composed of a sample absorption pad, a labeled reagent binding pad, a 0.22um microporous membrane, a coated reagent binding pad, and a hydrophilic porous membrane. The sample absorption pad, the labeled reagent binding pad, the coated reagent binding pad, the hydrophilic porous membrane, and the microporous membrane are arranged in sequence from front to back. The hydrophilic porous membrane is formed by clamping the hydrophilic surfaces of two layers of hydrophilic membranes facing each other, and maintaining a gap of 0.15mm extending forward by about 5mm. The solution of the hydrophilic porous membrane is drained out by the hydrophilic membranes clamping the chromatographic membranes above and below, and the solution directly flows into a solution outflow outlet arranged at the end of the bottom hydrophilic membrane, and the 0.22um microporous membrane is attached to the solution outflow outlet.

[0079] The labeling reagent binding pad in the immune lateral flow channel is solidified with colloidal gold particles with a particle size of about 40 nm labeled with FOB monoclonal antibody A, and the coating reagent binding pad is solidified with coated microspheres with a diameter of 1.5 um and FOB monoclonal antibody A fixedly coated on the surface. When the fecal sample solution is added to the detection device, the FOB protein flows in the immune lateral flow channel, and the solution will first react and combine with the colloidal gold particles labeled with FOB monoclonal antibody A, and then the solution will pass through the 0.22um filtration microporous membrane, and all impurity particles with a particle size larger than the pore size will be retained. The colloidal gold particle complex that has already reacted preliminarily will further react and combine with the coated microspheres corresponding to FOB monoclonal antibody B to form a reaction complex of two monoclonal antibodies sandwiching HCG protein. Subsequently, when passing through the 0.22um microporous membrane, the free HCG monoclonal antibody A-labeled colloidal gold particles with a particle size of about 40nm will pass through the microporous membrane and flow away, and the coated microspheres of the reaction complex and the free coated microspheres will be retained on the surface of the microporous membrane. These reaction complexes can be used to judge and analyze the concentration of HCG in the solution through the user's naked eyes or the matching colloidal gold quantitative analysis instrument.

[0080] In summary, the present invention adds a new functional structure, a microporous membrane, on the basis of immunochromatographic detection. On the one hand, the immunoreaction complex is captured by the microporous membrane using the dot immunocolloid gold permeation membrane method. On the other hand, the microporous membrane is used to intercept the microsphere reaction complex after sufficient mixing and incubation reaction in the lateral flow process. The reaction complex after sufficient mixing and incubation reaction in the lateral flow process is captured or physically intercepted by the microporous membrane. If it is physically intercepted, the appearance of the reaction complex no longer depends on the affinity of the ligand.

[0081] In the immunolateral flow and dot gold immunofiltration methods, the pore size of the microporous membrane is generally 0.45um, so the flow rate of the solution through the membrane is much slower. Therefore, immune reactions with weaker affinity can obtain suitable results in the filtration experiment. In the immune lateral flow and microsphere microporous membrane methods, the capture and retention of immune reaction complexes during the lateral flow process no longer depends on the affinity of the ligand. In the present invention, the coated reagent can be set on the microspheres suspended in the aqueous solution, and the microspheres are set on the solution flow channel. When the sample solution to be tested enters the lateral solution flow channel, the capture reagent and the labeling reagent are mixed and dissolved respectively, and the substance to be tested, the capture reagent and the labeling reagent in the sample solution are mixed and incubated and immunologically bound in the lateral flow of the solution to form an immune or ligand reaction complex on the surface of the microsphere. After a certain distance of immune directional flow, the components in the solution reach the microporous membrane in turn, and the components with a particle size smaller than the pore size of the microporous membrane pass through the microporous membrane and flow to the water-absorbing material such as filter paper; the diameter of the coated microspheres is larger than the pore size of the microporous membrane, and the free coated microspheres and the coated microspheres that form the reaction complex are retained by the microporous membrane at the outlet of the solution.

[0082] The above is only a preferred embodiment of the present invention. It should be noted that a person skilled in the art can make several improvements and modifications without departing from the principle of the present invention. These improvements and modifications should also be considered as the protection scope of the present invention.

Claims

1. A microporous membrane filtration detection device based on immune lateral flow, characterized in that: It includes an immune lateral flow channel and a microporous membrane with a pore size of less than 1.2um. The solution in the immune lateral flow channel directly flows into a solution outlet arranged at the end thereof. The microporous membrane is attached to the solution outlet to allow the solution to pass through and filter. The immune lateral flow channel includes a hydrophilic porous membrane capable of directional transmission of aqueous solution, wherein the hydrophilic porous membrane is selected from one or more of nitrocellulose membrane, glass fiber membrane, chemical fiber membrane and filter paper; the immune lateral flow channel also includes a void channel constructed by clamping the hydrophilic porous membrane by the hydrophilic surfaces of the upper and lower hydrophilic membranes, and the surface tension of the solution generated by the hydrophilic surface of the hydrophilic membrane is used as the power of liquid flow, and the solution flows from one end of the void channel to the other end; the sample absorption pad glass fiber membrane is used as the sample absorption pad and the reagent binding pad, the chemical fiber membrane is used as the hydrophilic membrane to guide the water channel, and the filter paper is used as the water absorbing material; The lower surface of the hydrophilic membrane attached to the microporous membrane in the immune lateral flow channel is coated with glue or adhesive, and the microspheres in the solution will not flow away from the gap between the hydrophilic membrane and the microporous membrane, but can only flow out from the microporous membrane attached at the gap of the hydrophilic membrane serving as the solution outflow outlet; At least one labeling reagent is arranged in the immune lateral flow channel, and a capture reagent is coated on the microporous membrane; the capture reagent and the labeling reagent are respectively provided with substances that are direct or indirect ligands to each other; The labeling reagent includes color particles or fluorescent substances that are directly visible to the naked eye; the color particles are selected from one or more of nano-colloidal gold, latex microspheres, carbon black particles, and other nanoparticles with color signals; the fluorescent substance is selected from one or more of fluorescent molecules and their microspheres, quantum dot particles and their microspheres, up-conversion luminescent particles and their microspheres, time-resolved fluorescent molecules and their microspheres, and fluorescent proteins.

2. The microporous membrane filtration detection device based on immune lateral flow according to claim 1, characterized in that: It also includes a detection device that can irradiate the reaction complex accumulated on the microporous membrane after the immune lateral flow with light of a specific wavelength and receive at least one wavelength spectrum signal reflected after being excited.

3. A microporous membrane filtration detection device based on immune lateral flow, characterized in that: It includes an immune lateral flow channel and a microporous membrane with uniform pore size in the range of 0.02-1.2um. The solution of the immune lateral flow channel flows to a solution outlet arranged at the end thereof. The microporous membrane is attached to the solution outlet for the solution to pass through and filter. The microporous membrane is hydrophilic. The immune lateral flow channel includes a hydrophilic porous membrane capable of directional transmission of aqueous solution, wherein the hydrophilic porous membrane is selected from one or more of nitrocellulose membrane, glass fiber membrane, chemical fiber membrane and filter paper; the immune lateral flow channel also includes a void channel constructed by clamping the hydrophilic porous membrane by the hydrophilic surfaces of the upper and lower hydrophilic membranes, and the surface tension of the solution generated by the hydrophilic surface of the hydrophilic membrane is used as the power of liquid flow, and the solution flows from one end of the void channel to the other end; the sample absorption pad glass fiber membrane is used as the sample absorption pad and the reagent binding pad, the chemical fiber membrane is used as the hydrophilic membrane to guide the water channel, and the filter paper is used as the water absorbing material; The lower surface of the hydrophilic membrane attached to the microporous membrane in the immune lateral flow channel is coated with glue or adhesive, and the microspheres in the solution will not flow away from the gap between the hydrophilic membrane and the microporous membrane, but can only flow out from the microporous membrane attached at the gap of the hydrophilic membrane serving as the solution outflow outlet; The immune lateral flow channel is provided with at least one coated microsphere with uniform diameter larger than the pore size of the microporous membrane, a capture reagent fixed on the surface of the microsphere, and a labeling reagent with a diameter smaller than the pore size of the microporous membrane; the capture reagent and the labeling reagent are respectively provided with substances that are direct or indirect ligands to each other.

4. The microporous membrane filtration detection device based on immune lateral flow according to claim 3, characterized in that: Two hydrophilic membranes are arranged at the end of the immune lateral flow channel, the hydrophilic surfaces of the two hydrophilic membranes clamp the upper and lower surfaces of the hydrophilic porous membrane, the lower surface of the hydrophilic membrane at the bottom of the two hydrophilic membranes is attached to the microporous membrane, and a notch is provided in the middle of the hydrophilic membrane attached to the upper surface of the microporous membrane as a solution outflow outlet; or, the hydrophilic surfaces of the two hydrophilic membranes clamp the left and right sides of the hydrophilic porous membrane, and a notch is provided in the middle of the bottom surface of the gap channel formed by the two hydrophilic membranes as a solution outflow outlet.

5. The microporous membrane filtration detection device based on immune lateral flow according to claim 4, characterized in that: The notch shapes arranged on the hydrophilic membrane at least include a gap shape with a maximum gap not exceeding 1.5 mm or an angle not exceeding 95 degrees.

6. The microporous membrane percolation detection device based on immune lateral flow according to claim 3, characterized in that: The lower surface of the microporous membrane is attached with a water-absorbing material, and the solution flowing into the gap of the hydrophilic membrane passes through the microporous membrane and is absorbed and transferred by the water-absorbing material.

7. The microporous membrane filtration detection device based on immune lateral flow according to claim 3, characterized in that: The labeling reagent includes color particles or fluorescent substances that are directly visible to the naked eye; the color particles are selected from one or more of nano-colloidal gold, latex microspheres, carbon black particles, and other nanoparticles with color signals; the fluorescent substance is selected from one or more of fluorescent molecules and their microspheres, quantum dot particles and their microspheres, up-conversion luminescent particles and their microspheres, time-resolved fluorescent molecules and their microspheres, and fluorescent proteins.

8. The microporous membrane filtration detection device based on immune lateral flow according to claim 7, characterized in that: It also includes a detection device that can irradiate the reaction complex trapped and accumulated on the surface of the microporous membrane after the immune lateral flow with light of a specific wavelength and receive at least one wavelength spectral signal reflected after being excited.

9. The microporous membrane filtration detection device based on immune lateral flow according to claim 1, characterized in that: When the coated microspheres and the microspheres formed by the reaction complexes with the labeling reagents are accumulated on the surface of the microporous membrane, the free labeling reagents can be carried by the solution through the pores between the accumulated microspheres and then flow out of the micropores of the microporous membrane again without forming any blocking residues.

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