Washing-free lateral flow chromatography fluorescence polarization immunoreaction analysis method

Abstract

The invention relates to the technical field of micro-fluidic chips, and discloses a test strip for lateral flow chromatography fluorescence polarization immunoreaction analysis, an OSTE porous medium is used as a substrate material, and the OSTE porous medium has a cross-linked micro-column structure; a reaction area and a conventional area are arranged; an antibody is fixed in the reaction area through a click chemical reaction; the fixation is carried out through active groups on the surface of the OSTE porous medium. The invention also discloses a preparation method of the test strip for lateral flow chromatography fluorescence polarization immunoreaction analysis, and also discloses a washing-free lateral flow chromatography fluorescence polarization immunoreaction analysis method. The method is easy and convenient to operate, rapid in reaction and high in sensitivity, fluorescence polarization signals can be obtained without washing the test strip, the fluorescence polarization signals detected by the test strip are strong, repeatability is high, quantification can be achieved, and the method is suitable for complex liquid samples and detection of various markers.

Description

Technical Field

[0001] The invention relates to the technical field of microfluidic chips, in particular to a washing-free lateral flow chromatography fluorescence polarization immunoreaction analysis method. Background Art

[0002] As the most representative instant detection technology, lateral flow chromatography immunoassay has made great progress in the past two decades and is widely used in instant detection of liquid samples. The immune reaction based on enzyme-linked immunosorbent assay (ELISA) is a common method for lateral flow chromatography immunoassay, but this method has poor repeatability and is difficult to quantify, and it is difficult to overcome the shortcomings of heterogeneous immune reactions, and the performance of immunoassay is limited. The existing high-sensitivity lateral flow chromatography test strips need to be washed and the fluorescent signal is obtained by fluorescence microscopy, such as the lateral flow chromatography immunoassay method in Development of a lateral flow strip membrane assay for rapid and sensitive detection of the SARS-CoV-2 (Analytical Chemistry, 2020, 92 (20): 14139-14144) by Yu S et al., which needs to be washed before scanning the test strip and reading the fluorescent signal.

[0003] The test strips using fluorescent polarization immune signals do not need to be washed and can be obtained through a polarization microscope. However, in the reading of polarization signals, traditional lateral flow chromatography test strips are mostly made of fibers, and the autofluorescence signal is too strong, so it will generate too much noise and it is difficult to obtain polarization signals. The Chinese patent application (CN113910505A, 2022-01-11) uses a new polymer OSTE to quickly prepare a microcolumn array. This microcolumn array can be used as a capillary pump to provide flow related to clinical testing, but its preparation requires laser cutting, molding and other steps, and mold lithography cannot be used to prepare high-precision three-dimensional structures. Summary of the invention

[0004] In order to solve the above technical problems, the present invention adopts the following technical solution: using OSTE porous medium as the base material and non-competitive immune reaction with fluorescence polarization signal as the reaction signal as the test principle, a test strip for lateral flow chromatography fluorescence polarization immunoreaction analysis is prepared to realize the quantitative analysis of multiple markers in liquid samples.

[0005] The invention provides a test strip for lateral flow chromatography fluorescence polarization immunoreaction analysis, which uses an OSTE porous medium as a base material. The OSTE porous medium has a cross-linked micro-column structure.

[0006] Furthermore, a reaction area and a conventional area are provided; the antibody is fixed to the reaction area by a click chemistry reaction; the fixation is performed by the thiol group on the surface of the OSTE porous medium; and the antibody is bound to an immunofluorescent group.

[0007] Furthermore, the OSTE porous medium is formed by the cross-linked microcolumn structure and the substrate sheet; the cross-linked microcolumn structure is formed by connecting array points on a plane parallel to the substrate sheet with adjacent array points; the array points are formed by microcolumns inclined to the substrate sheet; the number of adjacent array points includes any integer from 3 to 6.

[0008] Furthermore, the angle between the microcolumn and the substrate sheet is 15° to 75°, the thickness of the microcolumn is 200 microns to 10 millimeters, the diameter of the microcolumn is 50 to 200 microns, and the spacing between the microcolumns is 50 to 400 microns; the cross-linked microcolumn structure is formed by connecting an array point on a plane parallel to the substrate sheet with four adjacent array points.

[0009] Furthermore, in the reaction area, the diameter of the microcolumn is 100 microns, and the spacing between the microcolumns is 100 microns; in the conventional area, the diameter of the microcolumn is 150 microns, and the spacing between the microcolumns is 150 microns; the angle between the microcolumn and the substrate is 30°; and the thickness of the microcolumn is 200 microns.

[0010] Furthermore, a plurality of different antibodies are fixed at different positions of the reaction area through click chemistry reaction to form a plurality of reaction areas.

[0011] Furthermore, the preparation method of the cross-linked microcolumn structure includes curing the OSTE solution with ultraviolet light in multiple directions; the ratio of the number of thiol groups and vinyl groups in the OSTE solution is 1.4:1; the curing time is 3 minutes; the ultraviolet light is obtained by mirror reflection; the mirror is an aluminum mirror or a silicon wafer with aluminum coating on the surface.

[0012] The present invention also provides a method for preparing the above-mentioned test strip for lateral flow chromatography fluorescence polarization immunoreaction analysis, the method comprising:

[0013] Step 1) Preparation of lateral flow chromatography test strip: preparing the OSTE porous medium with the OSTE solution, and preparing the lateral flow chromatography test strip with the OSTE porous medium as the base material;

[0014] Step 2) Immobilization of the immunological reagent: combining the antibody with the immunofluorescent group to obtain a fluorescent antibody, and combining the fluorescent antibody with the lateral flow chromatography test strip described in step 1 to obtain the test strip for lateral flow chromatography fluorescence polarization immunoreaction analysis.

[0015] Furthermore, the method for preparing the test strip for lateral flow chromatography fluorescence polarization immunoreaction analysis comprises:

[0016] Step 1) Preparation of lateral flow chromatography test strips:

[0017] S1-1, firstly place the base sheet horizontally;

[0018] S1-2, adding thickness supports around the base sheet;

[0019] S1-3, dropping the OSTE solution onto the substrate;

[0020] S1-4, covering a mask having microholes on the thickness support;

[0021] S1-5, curing the OSTE solution in multiple directions with ultraviolet light to obtain the OSTE porous medium, wherein the angle between the ultraviolet light and the substrate is 15° to 75°;

[0022] S1-6, using the OSTE porous medium as a base material, cutting the OSTE porous medium to prepare a lateral flow chromatography test strip;

[0023] When the diameter of the micro-column is adjusted, the diameter of the micro-holes of the mask is adjusted, and when the spacing of the micro-columns is adjusted, the spacing of the micro-holes of the mask is adjusted;

[0024] Step 2) Immobilization of immunoreagents:

[0025] S2-1, first use a blocking solution to perform surface treatment on the reaction area of ​​the lateral flow chromatography test strip described in step 1;

[0026] S2-2, combining the antibody with the immunofluorescent group to obtain a fluorescent antibody, and then dripping the fluorescent antibody into the reaction area, drying, and obtaining a dried lateral flow chromatography test strip;

[0027] S2-3. Fix the fluorescent antibody on the dried lateral flow chromatography test strip using a click chemistry reaction to obtain the test strip for lateral flow chromatography fluorescence polarization immunoreaction analysis.

[0028] The present invention also provides a wash-free lateral flow chromatography fluorescence polarization immunoreaction analysis method, comprising the following steps: dropping a liquid sample containing a detection target onto any of the above-mentioned test strips for lateral flow chromatography fluorescence polarization immunoreaction analysis to obtain a target sample test strip, placing the target sample test strip into a fluorescence polarization signal measurement system, measuring the fluorescence polarization signal intensity of the detection target, and calculating the concentration of the detection target in the liquid sample.

[0029] Furthermore, the fluorescence polarization signal measurement system includes: an LED light source, an excitation filter, a liquid crystal layer, a polarization filter, a divergence filter and a CMOS sensor.

[0030] Furthermore, placing the target sample test strip into the fluorescence polarization signal measurement system specifically includes: placing the target sample test strip between the excitation filter and the liquid crystal layer.

[0031] Furthermore, the spacing between the micro-pillars is the shortest surface distance between a micro-pillar vertex of the array point and a micro-pillar vertex of an adjacent array point.

[0032] Furthermore, the substrate sheet is an OSTE substrate sheet.

[0033] Furthermore, after curing by ultraviolet light, the cross-linked micro-column structure and the base sheet are fixedly connected to form an OSTE porous medium.

[0034] Furthermore, the OSTE substrate can be conventionally prepared using existing techniques.

[0035] Furthermore, the antibodies are conventional protein antibodies, including vitamin antibodies, human chorionic gonadotropin, and C-reactive protein antibodies.

[0036] Furthermore, the click chemistry reaction for immobilizing antibodies involves addition reactions of carbon-carbon multiple bonds.

[0037] Furthermore, the click chemistry reaction for fixing the antibody is a Michael addition reaction.

[0038] Furthermore, the OSTE solution is prepared from three reagents: pentaerythritol tetrathioglycolate (thiol monomer), triallyl cyanurate (vinyl monomer) and photoinitiator Igr 819.

[0039] Furthermore, the mass ratio is thiol monomer: vinyl monomer: photoinitiator Igr819=13:7:0.02.

[0040] Furthermore, in step 1 of the above preparation method, after the UV light curing is completed, the OSTE porous medium is washed to remove the OSTE solution that has not been cured by the UV light.

[0041] Further, the thickness of the thickness support is the same as the thickness of the microcolumn.

[0042] Furthermore, the angle between the ultraviolet light and the substrate is the same as the angle between the microcolumns and the substrate.

[0043] Furthermore, the diameter of the microholes of the mask is equivalent to the diameter of the microcolumns, and the spacing between the microholes of the mask is equivalent to the spacing between the microcolumns.

[0044] Furthermore, the angle of the ultraviolet light is controlled by adjusting the angle of the aluminum mirror.

[0045] Furthermore, when the cross-linked microcolumn structure is formed by connecting an array point on a plane parallel to the substrate sheet with four adjacent array points, in step S1-5, the multiple directions are four directions, and the vertical projections of the angles of adjacent directions of ultraviolet light on the plane of the substrate sheet are all right angles.

[0046] Furthermore, in the preparation method of the present invention, the blocking solution is a 1% aqueous solution of gelatin, a 1% aqueous solution of bovine serum albumin or a 1% aqueous solution of polyvinyl pyrrolidone.

[0047] Furthermore, the wash-free lateral flow chromatography fluorescence polarization immunoreaction analysis method of the present invention is used to analyze multiple detection targets in a sample.

[0048] Furthermore, the principle of measuring the intensity of the fluorescence polarization signal of the detection target in the present invention is based on the twisted nematic mode of the liquid crystal display principle, and the liquid crystal layer is used to process the fluorescence signal diverged by the OSTE porous medium.

[0049] OSTE stands for off-stoichiometry thiol-enes. OSTE polymers are based on UV-curable thiol-ene chemical structures and utilize non-stoichiometric ratios to control their mechanical strength and surface chemical groups. The present invention provides a method for quantitative analysis of multiple markers in liquid samples using OSTE porous media as a substrate material in combination with non-competitive fluorescence polarization immunoreaction.

[0050] OSTE, with its weak autofluorescence signal, can effectively solve the noise problem of traditional lateral flow chromatography test strips, making polarization signals easier to obtain. Therefore, the method of using OSTE porous media as the base material to prepare test strips and combining fluorescence polarization immunoreaction in the present invention can effectively solve the problems of poor repeatability and difficulty in quantification in current lateral flow chromatography immunoassays, and can read fluorescence polarization signals without washing the test strips, which helps to improve the performance and efficiency of lateral flow chromatography immunoassays. Direct photolithography of OSTE can prepare high-precision three-dimensional structures; at the same time, by building a fluorescence polarization signal measurement system, the difficulty of reading polarization signals on porous media is overcome, so that polarization signals can be read more conveniently.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] 1. The present invention applies fluorescence polarization signals to lateral flow chromatography immunoreaction for the first time, which is simple to operate, rapid in response and highly sensitive. Moreover, compared with traditional lateral flow chromatography test strips, the present invention can obtain lateral flow chromatography immunoassay results without washing the test strips.

[0053] 2. The autofluorescence signal of the OSTE porous medium of the present invention is low, and the fluorescence polarization signal detected by the lateral flow chromatography test strip made of this as the base material has low noise, strong fluorescence polarization signal, high repeatability and quantification; at the same time, the antibody protein can be fixed to the surface group of the OSTE porous medium of the present invention by using click chemistry reaction, which is suitable for complex liquid samples and simultaneous detection of multiple markers.

[0054] 3. Compared with the prior art for preparing OSTE porous media, the present invention uses ultraviolet photolithography to prepare high-precision three-dimensional structures. The test strips prepared with this as the base material can control the signal intensity by adjusting the specific surface area, and the microstructure of the test strips is regular and uniform, which is conducive to enhancing the controllability of fluorescence polarization signal detection.

[0055] 4. The fluorescence polarization signal measurement system designed in the present invention makes the signal easier to read and is small in size, easy to carry and low in cost, further improving the practicality of the analytical method of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 Schematic diagram of the preparation of the OSTE porous medium (a) and the lateral flow chromatography test strip (b) of the present invention;

[0057] Figure 2 Schematic diagram of the OSTE porous medium in the present invention having different regions (a), the principle of OSTE porous medium modified with antibodies (b), the test strip having multiple reaction regions (c), and the test strip having sample added for detection (d);

[0058] Figure 3 Schematic diagram of the design of the fluorescence polarization signal measurement system in the present invention;

[0059] Figure 4 Schematic diagram of the fluorescence polarization signal measurement principle in the present invention;

[0060] Figure 5 This is a schematic diagram of the non-competitive fluorescence polarization reaction in the present invention;

[0061] Figure 6 This is a schematic diagram showing the effect of the binding of target 1 to antibody 1 on the molecular weight of antibody 1 in the present invention;

[0062] Figure 7 A schematic diagram of a lateral flow chromatography immunoassay test strip for testing multiple vitamins in the present invention;

[0063] Figure 8 This is a schematic diagram of the lateral flow chromatography steps in Comparative Example 1;

[0064] Fig. 9 This is the SEM structure diagram of the OSTE porous medium in Example 1. DETAILED DESCRIPTION

[0065] To make the purpose, technical scheme and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with specific examples, comparative examples and accompanying drawings. Unless otherwise specified, the reagents and materials used in the following examples and comparative examples are conventional commercial products.

[0066] Example 1 Preparation of lateral flow chromatography test strip using OSTE porous medium as base material

[0067] 1. Preparation of test strips:

[0068] The following steps 1 to 5 are to prepare the OSTE porous medium with cross-linked micro-column structure by UV photolithography. The preparation schematic diagram is shown in Figure 1 (a).

[0069] Step 1: Place the OSTE substrate horizontally.

[0070] Step 2: Add thickness support around the base sheet, and the thickness of the thickness support is 200 microns.

[0071] Step 3, then add 3 to 5 ml of viscous, transparent OSTE solution (the ratio of the number of thiol groups to vinyl groups is 1.4:1) onto the substrate. The OSTE solution is prepared from three reagents: pentaerythritol tetrathioacetate (thiol monomer), triallyl cyanurate (vinyl monomer) and photoinitiator Igr819. The mass ratio is thiol monomer: vinyl monomer: photoinitiator Igr819 = 13:7:0.02.

[0072] Step 4: Cover the thickness support with a mask (chrome plate) of 10.2 x 10.2 cm so that the mask fits tightly against the thickness support. The diameter of the micro-columns is controlled by adjusting the micro-hole diameter of the mask. The spacing of the micro-columns is controlled by adjusting the micro-hole spacing of the mask: the micro-hole diameter of the reaction area is set to 100 microns, and the micro-hole spacing is set to 100 microns. The micro-hole diameter of the conventional area is set to 150 microns, and the micro-hole spacing is set to 150 microns.

[0073] Step 5. Place aluminum mirrors with four sides tilted at a certain angle around the container for ultraviolet light reflection. The reflected tilted ultraviolet light irradiates the OSTE solution from four directions; the ultraviolet light irradiates and cures the OSTE solution through the mask for 3 minutes. In the four irradiated directions, the vertical projections of the angles of adjacent directions of ultraviolet light on the plane of the substrate are all right angles. The angle between the ultraviolet light and the substrate is 30°. The irradiation angle of the ultraviolet light is controlled by adjusting the angle of the aluminum mirror; finally, an OSTE porous medium with a cross-linked microcolumn structure is obtained, and the length and width are the same as those of the mask.

[0074] The specific specifications of the obtained OSTE porous medium are as follows: the thickness of the microcolumn is 200 microns, the diameter of the microcolumn is 100 microns in the reaction area and 150 microns in the conventional area, the spacing between the microcolumns is 100 microns in the reaction area and 150 microns in the conventional area, and the angle between the microcolumn and the substrate is 30°; since the denser the structure and the larger the surface area of ​​the reaction area, the stronger the signal, the microcolumn spacing and microcolumn diameter in the reaction area are set smaller than those in the conventional area, which can achieve the effect of enhancing the signal.

[0075] The SEM structure of the reaction area in the obtained OSTE porous medium is shown in Fig. 9 As shown, the diameter of the micro-pillars is 100 μm and the spacing between the micro-pillars is 100 μm.

[0076] Step 6: Figure 1 As shown in (b), the OSTE porous medium prepared in step 5 is cut to make a lateral flow chromatography test strip.

[0077] 2. Fixation of immunoreagents:

[0078] Step 1: Take the test strip obtained in step 1 and use a blocking solution to treat the surface of the reaction area of ​​the test strip to eliminate the interference of nonspecific adsorption on the detection signal; the reaction area and the conventional area are as follows: Figure 2 As shown in (a), the conventional area (1.5x) and the reaction area (1x) indicate that the diameter of the microcolumns in the conventional area and the spacing between the microcolumns are 1.5 times that of the reaction area.

[0079] Step 2: Combine the multivitamin antibodies with the corresponding immunofluorescent groups, then take 1 to 3 microliters of the antibody solution and drop it into the corresponding reaction area. Allow the test strip to dry in a constant temperature and humidity environment (temperature: 25 degrees Celsius, humidity: 50%) and without wind. After 12 hours of drying, the antibodies are deposited in the corresponding reaction area.

[0080] Step 3: Take the test strip with deposited antibodies obtained in step 2 and irradiate it with ultraviolet light (12 mw / cm 2 ) After 60s, washing is performed to obtain a test strip for lateral flow chromatography fluorescence polarization immunoreaction analysis; Figure 2 As shown in (b), the OSTE surface carries an active thiol group, and the M group is a maleimide group, which is the linking group of the antibody. The click chemistry Michael addition reaction is used to react and connect the M group and the surface group of the OSTE substrate, thereby fixing the antibody on the surface of the OSTE porous medium.

[0081] Finally Figure 2 As shown in (c), different vitamin antibodies are fixed at different positions of the test strip obtained in step 1 to set up multiple reaction areas, thereby obtaining a test strip for lateral flow chromatography fluorescence polarization immunoreaction analysis, and the target to be tested is multiple vitamins.

[0082] Example 2: Measuring the fluorescence polarization signals of various targets using the test strip prepared in Example 1

[0083] 1. Design the measurement system:

[0084] like Figure 3 The fluorescence polarization signal measurement system is designed as shown in the figure, which is mainly composed of an LED light source, an excitation filter, a liquid crystal layer, a polarization filter, a divergence filter and a CMOS sensor.

[0085] like Figure 4 As shown in the figure, the measurement principle of this system is based on the twisted nematic mode of the liquid crystal display principle, and the liquid crystal layer is used to process the fluorescence signal emitted by the OSTE porous medium. When the liquid crystal layer is not connected, the sensor measures the fluorescence polarization signal intensity I of the sample parallel to the excitation light plane. ∥ When the liquid crystal layer is in the on state, the sensor measures the fluorescence polarization signal intensity I of the sample perpendicular to the excitation light plane ⊥ .

[0086] Assume P is the fluorescence polarization signal intensity of the sample, according to the formula P = (I ∥ -I ⊥ )*(I ∥ +I ⊥ ), the fluorescence polarization signal intensity can be calculated.

[0087] 2. Signal detection: Fluorescence polarization immunoreaction is used to detect liquid samples in combination with the above-mentioned fluorescence polarization signal measurement system. The specific steps are as follows:

[0088] Step 1: drip a liquid sample containing multiple vitamins as the target to be tested onto the test strip for lateral flow chromatography fluorescence polarization immunoreaction analysis prepared in Example 1. Specifically, drip the sample onto the test strip. Figure 2 (c) The sample pad was subsequently tested for multiple vitamins.

[0089] like Figure 2 As shown in (d), when the liquid sample arrives at different reaction areas in sequence according to the flow direction, the various targets in the sample bind to the antibodies fixed in the corresponding reaction areas. Specifically, when the liquid sample arrives at the vitamin A reaction area, vitamin A binds to the vitamin A antibody in the reaction area; and when the liquid sample arrives at the vitamin B reaction area, vitamin B binds to the vitamin B antibody in the reaction area, and so on. Figure 7 shown.

[0090] The fluorescence polarization changes before and after the antibody binds to the target are as follows Figure 5-6 As shown, the binding of the vitamin to be tested and the corresponding vitamin antibody will affect the molecular weight of the antibody, thereby affecting the strength of the subsequent fluorescence polarization signal.

[0091] Step 2: Then place the test strip with the liquid sample added Figure 4 The light source is turned on between the excitation filter and the liquid crystal layer of the fluorescence polarization signal measurement system to measure the changes in the fluorescence polarization signal in different reaction areas. The fluorescence polarization signal intensity of each vitamin to be tested is calculated according to the fluorescence polarization signal intensity (P) formula; the concentration of the vitamin to be tested in the liquid sample is positively correlated with the corresponding fluorescence polarization signal intensity.

[0092] The detection result is that the fluorescence polarization signal P measured in Example 2 is about 30,000 points. In addition, the autofluorescence signal of the OSTE porous medium of the test strip substrate used in Example 2 is measured by fluorescence microscope and is about 500 points.

[0093] Comparative Example 1

[0094] Lateral flow immunoassay combined with ordinary fluorescence detection uses traditional cellulose test strips. The test strips need to be washed before scanning and the fluorescence signal is obtained through a fluorescence microscope. The steps are shown in Figure 8 .

[0095] Comparative Example 2

[0096] The autofluorescence signal of the cellulose substrate of the traditional cellulose test strip, when not washed, was measured by fluorescence microscope to be about 20,000 points. The results show that the autofluorescence signal of the traditional test strip is too strong, which produces a lot of noise and cannot obtain the fluorescence polarization signal of the immune response. By comparison, it can be seen that the OSTE porous medium (autofluorescence signal of about 500 points) of the test strip substrate of Example 2 of the present invention has made significant progress in reducing the autofluorescence signal compared with the cellulose substrate of the traditional test strip.

[0097] Comparative Example 3

[0098] The lateral flow immunoassay was combined with fluorescence polarization detection. The OSTE upright microcolumn array porous medium was prepared according to the mold photolithography method of the Chinese patent application (CN113910505A, 2022-01-11). The test strip was prepared according to the method of step 2 in Example 1 of the present invention, and the fluorescence polarization signal was read by the detection method of Example 2. The detection result was that the fluorescence polarization signal P of Comparative Example 3 was about 7500 points. In addition, the autofluorescence signal of the OSTE porous medium on the base of the test strip of Comparative Example 3 was measured to be about 400 points.

[0099] Although the test strip with the OSTE upright micro-pillar array porous medium as the substrate in comparative example 3 has a much lower autofluorescence signal than the traditional cellulose substrate in comparative example 2, the above comparison results show that the fluorescence polarization signal intensity of Example 2 of the present invention (fluorescence polarization signal is about 30,000 points) is significantly improved compared with the test strip of comparative example 3. It can be seen that the application of fluorescence polarization on the OSTE porous medium with a cross-linked micro-pillar structure in the present invention is better than the application of fluorescence polarization on the OSTE porous medium with an upright micro-pillar array.

[0100] The above disclosure is only the preferred embodiment of the present invention, which cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A test strip for lateral flow chromatography fluorescence polarization immunoreaction analysis, characterized in that: An OSTE porous medium is used as a base material, and the OSTE porous medium has a cross-linked micro-column structure.

2. The test strip for lateral flow chromatography fluorescence polarization immunoreaction analysis according to claim 1, characterized in that: A reaction area and a conventional area are provided; the antibody is fixed on the reaction area through a click chemical reaction; the fixation is performed through the thiol group on the surface of the OSTE porous medium; and the antibody is bound to an immunofluorescent group.

3. The test strip for lateral flow chromatography fluorescence polarization immunoreaction analysis according to claim 2, characterized in that: The OSTE porous medium is formed by the cross-linked microcolumn structure and the substrate; the cross-linked microcolumn structure is formed by connecting array points on a plane parallel to the substrate with adjacent array points; the array points are formed by microcolumns inclined to the substrate; the number of adjacent array points includes any integer from 3 to 6.

4. The test strip for lateral flow chromatography fluorescence polarization immunoreaction analysis according to claim 3, characterized in that: The angle between the microcolumn and the substrate sheet is 15° to 75°, the thickness of the microcolumn is 200 microns to 10 mm, the diameter of the microcolumn is 50 to 200 microns, and the spacing between the microcolumns is 50 to 400 microns; the cross-linked microcolumn structure is formed by connecting an array point on a plane parallel to the substrate sheet with four adjacent array points.

5. The test strip for lateral flow chromatography fluorescence polarization immunoreaction analysis according to claim 3, characterized in that: In the reaction area, the diameter of the microcolumn is 100 microns, and the spacing between the microcolumns is 100 microns; in the conventional area, the diameter of the microcolumn is 150 microns, and the spacing between the microcolumns is 150 microns; the angle between the microcolumn and the substrate is 30°; the thickness of the microcolumn is 200 microns.

6. The test strip for lateral flow chromatography fluorescence polarization immunoreaction analysis according to claim 2, characterized in that: A plurality of different antibodies are fixed at different positions of the reaction area through click chemistry reaction to form a plurality of reaction areas.

7. The test strip for lateral flow chromatography fluorescence polarization immunoreaction analysis according to claim 1, characterized in that: The preparation method of the cross-linked microcolumn structure includes curing an OSTE solution by multi-directional irradiation with ultraviolet light; the ratio of the number of thiol groups and vinyl groups in the OSTE solution is 1.4:1; the irradiation curing time is 3 minutes; the ultraviolet light is obtained by mirror reflection; the mirror is an aluminum mirror or a silicon wafer with aluminum coating on the surface.

8. A method for preparing a test strip for lateral flow chromatography fluorescence polarization immunoreaction analysis according to claim 3, characterized in that: include: Step 1) Preparation of lateral flow chromatography test strip: preparing the OSTE porous medium with an OSTE solution, and preparing the lateral flow chromatography test strip with the OSTE porous medium as a base material; Step 2) Immobilization of the immunoreagent: combining the antibody with the immunofluorescent group to obtain a fluorescent antibody, and combining the fluorescent antibody with the lateral flow chromatography test strip in step 1 to obtain the test strip for lateral flow chromatography fluorescence polarization immunoreaction analysis.

9. The preparation method according to claim 8, characterized in that: include: Step 1) Preparation of lateral flow chromatography test strips: S1-1, firstly place the base sheet horizontally; S1-2, adding thickness supports around the base sheet; S1-3, dropping the OSTE solution onto the substrate; S1-4, covering a mask having microholes on the thickness support; S1-5, curing the OSTE solution in multiple directions with ultraviolet light to obtain the OSTE porous medium, wherein the angle between the ultraviolet light and the substrate is 15° to 75°; S1-6, using the OSTE porous medium as a base material, cutting the OSTE porous medium to prepare a lateral flow chromatography test strip; When the diameter of the micro-column is adjusted, the diameter of the micro-holes of the mask is adjusted, and when the spacing of the micro-columns is adjusted, the spacing of the micro-holes of the mask is adjusted; Step 2) Immobilization of immunoreagents: S2-1, first use a blocking solution to perform surface treatment on the reaction area of ​​the lateral flow chromatography test strip described in step 1; S2-2, combining the antibody with the immunofluorescent group to obtain the fluorescent antibody, and then dripping the fluorescent antibody into the reaction area, drying, and obtaining a dried lateral flow chromatography test strip; S2-3. Fix the fluorescent antibody on the dried lateral flow chromatography test strip using a click chemistry reaction to obtain the test strip for lateral flow chromatography fluorescence polarization immunoreaction analysis.

10. A wash-free lateral flow chromatography fluorescence polarization immunoreaction analysis method, characterized in that: The method comprises the following steps: dripping a liquid sample containing a detection target onto the test strip for lateral flow chromatography fluorescence polarization immunoreaction analysis as claimed in claim 1 to obtain a target sample test strip, placing the target sample test strip into a fluorescence polarization signal measurement system, measuring the fluorescence polarization signal intensity of the detection target, and calculating the concentration of the detection target in the liquid sample; The fluorescence polarization signal measurement system comprises: an LED light source, an excitation filter, a liquid crystal layer, a polarization filter, a divergence filter and a CMOS sensor.

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