Molecularly imprinted polymer, preparation method and application thereof, and detection chip

Abstract

The invention provides a molecularly imprinted polymer, a preparation method and application thereof, and a detection chip, and relates to the technical field of biomedicine, the molecularly imprinted polymer comprises: a matrix, the surface of which is provided with a plurality of first antigen sites and a plurality of antibody sites; the plurality of fluorescent antibodies are specifically combined with the matrix at the antibody sites, and the fluorescent antibodies are used for generating fluorescent signals; wherein each fluorescent antibody is provided with a second antigen site, and the first antigen site and the second antigen site are used for carrying out specific binding with a target antigen.

Description

Technical Field

[0001] Embodiments of the present application relate to the field of biomedical technologies, and in particular, to a molecularly imprinted polymer, a preparation method and application thereof, and a detection chip. Background Art

[0002] Microfluidics is a biomedical technology that integrates basic operation units such as sample preparation, reaction, separation, and detection in the process of biological, chemical, and medical analysis onto a single chip to automatically complete the entire analysis process. The principle of an immunological analysis microfluidic chip is to utilize the specific binding of antigen and antibody to complete the detection of the analyte. Microfluidic chips have advantages such as shortening the reaction time, reducing reagent consumption, simple operation, integration, automation, and portability, and are new types of immunodiagnostic consumables. Moreover, when using a microfluidic chip for detection, the required sample volume is low, and multiple relevant biological information or disease detection results can be obtained through a single detection, which conforms to the development trend of in vitro diagnosis.

[0003] However, in existing microfluidic chips, when each capture antibody binds to an antigen carrier, since there is only one fluorescent antibody on each antigen carrier bound to the antigen, each capture antibody can only correspond to one fluorescent antibody. This results in a relatively small number of fluorescent antibodies corresponding to the capture antibody during the detection process, thereby leading to poor fluorescence signal intensity of the fluorescent antibody and low detection sensitivity. Therefore, providing an antigen carrier that can amplify the fluorescence signal intensity has become an urgent problem to be solved in the current field. Summary of the Invention

[0004] Embodiments of the present application aim to provide a preparation method and application of a molecularly imprinted polymer, aiming to provide an antigen carrier that can amplify the fluorescence signal intensity.

[0005] In a first aspect of the embodiments of the present application, a molecularly imprinted polymer is provided, and the molecularly imprinted polymer includes:

[0006] A matrix, on the surface of which a plurality of first antigen sites and a plurality of antibody sites are provided;

[0007] A plurality of fluorescent antibodies, which specifically bind to the matrix at the antibody sites, and the fluorescent antibodies are used to generate fluorescence signals;

[0008] Wherein, a second antigen site is provided on each of the fluorescent antibodies, and the first antigen site and the second antigen site are used to specifically bind to a target antigen.

[0009] In an alternative embodiment, the antibody site is a specific cavity corresponding to the fluorescent antibody on the surface of the substrate, and the fluorescent antibody forms a connection with the substrate at the antibody site through intermolecular forces.

[0010] In an alternative embodiment, the first antigen site is a specific cavity corresponding to the target antigen on the surface of the substrate.

[0011] A second aspect of the embodiments of the present application provides a detection chip, which includes a detection substrate and a cover plate located on one side of the detection substrate;

[0012] The cover plate includes a sample inlet for injecting a sample;

[0013] The detection substrate includes a first fixing area, which is communicated with the sample inlet. The molecularly imprinted polymer according to any one of claims 1 to 3 is pre-set in the first fixing area, and the first fixing area is used to specifically bind to the target antigen in the sample based on the molecularly imprinted polymer.

[0014] In an alternative embodiment, the detection substrate further includes:

[0015] A second fixing area, which is communicated with the side of the first fixing area away from the sample inlet. A capture antibody is grafted in the second fixing area, and the second fixing area is used to capture the molecularly imprinted polymer that has bound the target antigen based on the capture antibody.

[0016] In an alternative embodiment, the first fixing area includes:

[0017] A first liquid inlet part arranged on the side close to the sample inlet and a first fixing part arranged on the side away from the sample inlet. The width of the first liquid inlet part gradually increases in a first direction, and the first direction is the arrangement direction of the sample inlet and the first fixing area;

[0018] A first diversion part, which is arranged at the edge of the first liquid inlet part and is used to make the sample flow forward along the first diversion part to the first liquid inlet part. The thickness of the first diversion part is greater than the thickness of the first liquid inlet part and the first fixing part.

[0019] In an alternative embodiment, the shape of the first liquid inlet part is semi-circular, and the shape of the first diversion part is a semi-circular ring arranged at the edge of the first liquid inlet part.

[0020] A third aspect of the embodiments of the present application provides a method for preparing a molecularly imprinted polymer, and the method includes:

[0021] Provide a matrix, on the surface of which there are provided a plurality of first antigen sites and a plurality of antibody sites;

[0022] Bind a plurality of fluorescent antibodies to the matrix, the fluorescent antibodies specifically binding to the matrix at the antibody sites, the fluorescent antibodies being used to generate fluorescent signals; wherein, each of the fluorescent antibodies is provided with a second antigen site, and the first antigen site and the second antigen site are used to specifically bind to a target antigen.

[0023] In an optional embodiment, the providing of the matrix includes:

[0024] Mix a first template molecule, a second template molecule and a functional monomer to obtain a first solution, the first template molecule being the target antigen, and the second template molecule being the fluorescent antibody;

[0025] Add a crosslinking agent to the first solution to cause pre-polymerization of the first template molecule, the second template molecule and the functional monomer in the first solution to obtain a second solution;

[0026] Add an initiator to the second solution to cause a covalent bond to be formed between the functional monomer and the crosslinking agent in the second solution to obtain the first product;

[0027] Remove the first template molecule and the second template molecule from the first product to form the plurality of first antigen sites and the plurality of antibody sites on the surface of the first product, thereby obtaining the matrix.

[0028] The fourth aspect of the embodiments of the present application provides an application of the molecularly imprinted polymer according to any one of the first aspects in the detection of a target antigen in a sample.

[0029] Advantageous effects:

[0030] The present application provides a molecularly imprinted polymer, a preparation method and an application thereof, and a detection chip. The molecularly imprinted polymer includes: a matrix, on the surface of which there are provided a plurality of first antigen sites and a plurality of antibody sites; a plurality of fluorescent antibodies, the fluorescent antibodies specifically binding to the matrix at the antibody sites, the fluorescent antibodies being used to generate fluorescent signals; wherein, each of the fluorescent antibodies is provided with a second antigen site, and the first antigen site and the second antigen site are used to specifically bind to a target antigen. The molecularly imprinted polymer provided by the present application contains a plurality of fluorescent antibodies. After the first antigen site and the second antigen site on the molecularly imprinted polymer bind to the target antigen in the sample, the fluorescence signal intensity when the target antigen is detected can be effectively amplified by the plurality of fluorescent antibodies, thereby improving the detection sensitivity. Description of the Drawings

[0031] To more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings required for the description of the embodiments of the present application. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0032] Figure 1 It is a schematic structural diagram of a molecularly imprinted polymer proposed in an embodiment of the present application;

[0033] Figure 2 It is a schematic structural diagram of a substrate proposed in an embodiment of the present application;

[0034] Figure 3 It is a schematic structural diagram of a detection chip proposed in an embodiment of the present application;

[0035] Figure 4 It is an enlarged schematic structural diagram of a first fixing area proposed in an embodiment of the present application;

[0036] Figure 5 It is a schematic diagram of the detection principle of a detection chip applied to a target antigen proposed in an embodiment of the present application;

[0037] Figure 6 It is a flowchart of a preparation method of a molecularly imprinted polymer proposed in an embodiment of the present application.

[0038] Explanation of reference numerals: 1, substrate; 2, first antigen site; 3, antibody site; 4, fluorescent antibody; 5, second antigen site; 6, target antigen; 7, capture antibody; 11, sample inlet; 12, first mixing area; 13, first fixing area; 131, first liquid inlet part; 132, first fixing part; 133, first diversion part; 14, second mixing area; 15, second fixing area; 16, waste liquid pool; 17, sample outlet. Specific embodiments

[0039] The following will clearly and completely describe the technical solutions in the embodiments of the present application with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present application.

[0040] In the accompanying drawings, for the sake of clarity, the sizes of components, the thicknesses of layers, or regions may sometimes be exaggerated. Therefore, any implementation of the present disclosure is not necessarily limited to the dimensions shown in the figures, and the shapes and sizes of components in the drawings do not reflect the true proportions. In addition, the drawings schematically show ideal examples, and any implementation of the present disclosure is not limited to the shapes or values shown in the drawings, etc.

[0041] Microfluidics is a biomedical technology that integrates basic operation units such as sample preparation, reaction, separation, and detection in the processes of biological, chemical, and medical analysis onto a single chip to automatically complete the entire analysis process. The principle of the immunological analysis microfluidic chip is to utilize the specific binding of antigen and antibody to complete the detection of the analyte. Microfluidic chips have advantages such as shortening the reaction time, reducing reagent consumption, being easy to operate, being integrated, automated, and portable, and are new types of immunodiagnostic consumables. Moreover, when using a microfluidic chip for detection, the required sample volume is low, and multiple relevant biological information or disease detection results can be obtained through a single detection, which conforms to the development trend of in vitro diagnosis.

[0042] However, existing antigen detections usually adopt the double-antibody sandwich method for detection. Among them, when each capture antibody in the microfluidic chip binds to the antigen carrier, since there is only one fluorescent antibody on each antigen carrier bound with the antigen, each capture antibody can only correspond to one fluorescent antibody. This results in a relatively small number of fluorescent antibodies corresponding to the capture antibodies on the detection chip during the detection process using the double-antibody sandwich method, thereby leading to a poor fluorescence signal intensity of the fluorescent antibodies and a low detection sensitivity.

[0043] In view of this, an embodiment of the present application proposes a molecularly imprinted polymer. Figure 1 The following shows a schematic structural diagram of a molecularly imprinted polymer proposed in an embodiment of the present application, as Figure 1 shown, the molecularly imprinted polymer includes: a matrix 1; a plurality of fluorescent antibodies 4, the fluorescent antibodies 4 being used to generate fluorescence signals; wherein, a second antigen site 5 is provided on each of the fluorescent antibodies 4, and the first antigen site 2 and the second antigen site 5 are used to specifically bind to a target antigen.

[0044] In some alternative embodiments, Figure 2 The following shows a schematic structural diagram of a matrix proposed in an embodiment of the present application, as Figure 2As shown, the matrix 1 is formed by covalently connecting functional monomers and crosslinking agents. Multiple first antigen sites 2 and multiple antibody sites 3 are provided on the surface of the matrix 1. Among them, the multiple first antigen sites 2 are located on the surface of the matrix 1, and the spatial configuration of the first antigen sites 2 on the matrix 1 corresponds to the spatial structure of the target antigen, so that the first antigen sites 2 can only specifically bind to the target antigen; multiple antibody sites 3, the multiple antibody sites 3 are located on the surface of the matrix 1, and the spatial configuration of the antibody sites 3 on the matrix 1 corresponds to the spatial structure of the fluorescent antibody, so that the antibody sites 3 can only specifically bind to the fluorescent antibody.

[0045] In the embodiment of the present application, the molecularly imprinted polymer includes multiple fluorescent antibodies 4. The fluorescent antibodies 4 specifically bind to the matrix 1 at the antibody sites 3. The fluorescent antibodies 4 are used to generate fluorescent signals to achieve qualitative detection of the target antigen. Among them, a second antigen site 5 is provided on each fluorescent antibody 4. The first antigen site 2 and the second antigen site 5 are used to specifically bind to the target antigen.

[0046] The sites on the molecularly imprinted polymer provided in the embodiment of the present application that specifically bind to the target antigen include multiple first antigen sites 2 and second antigen sites 5 provided on the fluorescent antibody 4, so that the specific binding efficiency of the molecularly imprinted polymer provided in the embodiment of the present application to the target antigen is improved, and more target antigens in the sample can be bound, thereby improving the accuracy and sensitivity of the detection of the target antigen in the sample. On the other hand, since multiple fluorescent antibodies 4 corresponding to multiple antibody sites 3 are provided in the molecularly imprinted polymer provided in the embodiment of the present application, when the molecularly imprinted polymer is used as an antigen carrier and binds to the target antigen through the first antigen site 2 and / or the second antigen site 5, stronger fluorescent signals can be jointly generated by the multiple fluorescent antibodies 4 on each molecularly imprinted polymer, thereby amplifying the fluorescence intensity of each molecularly imprinted polymer and effectively improving the sensitivity of the detection of the target antigen in the sample.

[0047] Specifically, the antibody site 3 is a specific cavity corresponding to the fluorescent antibody on the surface of the matrix 1. The fluorescent antibody 4 forms a connection with the matrix 1 at the antibody site 3 through intermolecular forces (such as hydrogen bonds, etc.). Among them, the specific cavity corresponding to the fluorescent antibody 4 is a cavity formed with a template molecule based on the fluorescent antibody 4, and the spatial configuration of the antibody site 3 corresponds to the spatial structure of the template molecule of the fluorescent antibody 4. The first antigen site 2 is a specific cavity corresponding to the target antigen on the surface of the matrix 1. The specific cavity corresponding to the target antigen is a cavity formed with a template molecule based on the target antigen, and the spatial configuration of the first antigen site 2 corresponds to the spatial structure of the template molecule of the target antigen.

[0048] The present application provides a molecularly imprinted polymer, which includes: a matrix, and a plurality of first antigen sites and a plurality of antibody sites are arranged on the surface of the matrix; a plurality of fluorescent antibodies, and the fluorescent antibodies specifically bind to the matrix at the antibody sites, and the fluorescent antibodies are used to generate fluorescent signals; wherein, a second antigen site is arranged on each of the fluorescent antibodies, and the first antigen site and the second antigen site are used to specifically bind to a target antigen. The molecularly imprinted polymer provided by the present application contains a plurality of fluorescent antibodies. After the first antigen site and the second antigen site on the molecularly imprinted polymer bind to the target antigen in the sample, the fluorescent signal intensity when the target antigen is detected can be effectively amplified by the plurality of fluorescent antibodies, thereby improving the detection sensitivity.

[0049] Based on the same inventive concept, an embodiment of the present application provides a detection chip. Figure 3 The structural schematic diagram of a detection chip proposed in an embodiment of the present application is shown. As Figure 3 shown, the detection chip includes: a detection substrate and a cover plate located on one side of the detection substrate; the cover plate includes a sample inlet 11, and the sample inlet 11 is used for injecting a sample; the detection substrate includes a first fixing area 13, the first fixing area 13 is communicated with the sample inlet 11, and the molecularly imprinted polymer described in the embodiment of the present application is pre-set inside the first fixing area 13. In the first fixing area 13, the molecularly imprinted polymer specifically binds to the target antigen in the sample through the first antigen site 2 and the second antigen site 5 on the fluorescent antibody 4.

[0050] In some optional embodiments, Figure 4 The structural enlarged schematic diagram of a first fixing area proposed in an embodiment of the present application is shown. As Figure 4As shown, the first fixing area 13 includes: a first liquid inlet part 131 arranged on one side close to the sample inlet 11 and a first fixing part 132 on the side far from the sample inlet 11. The width of the first liquid inlet part 131 gradually increases in the first direction, and the first direction is the arrangement direction of the sample inlet 11 and the first fixing area 13; a first diversion part 133 is arranged at the edge of the first liquid inlet part 131 and is used to make the sample flow forward along the first diversion part 133 to the first liquid inlet part 131. Among them, the thickness of the first diversion part 133 is greater than the thicknesses of the first liquid inlet part 131 and the first fixing part 132, so that the first diversion part 133 and the first liquid inlet part 131 form a stepped structure. It should be noted that in the embodiments of the present application, the width mentioned refers to the dimension in the direction perpendicular to the main direction of the liquid in the detection chip, and the thickness refers to the dimension in the direction perpendicular to the upper and lower flat surfaces of the detection chip.

[0051] The sample first enters the first diversion part 133 of the first fixing area 13 through the sample inlet 11. Under the action of capillary action, the sample extends and diffuses along the edge of the first diversion part 133. After the sample liquid diffuses in the first diversion part 133, it flows to the first liquid inlet part 131, so as to realize the uniform sampling of the sample liquid based on the relatively wide first diversion part 133, thereby improving the sampling stability of the sample liquid in the first fixing area 13, reducing the generation of bubbles, enabling the molecularly imprinted polymer in the first fixing area 13 to effectively bind to the target antigen in the sample, and improving the detection sensitivity.

[0052] In some alternative embodiments, the shape of the first liquid inlet part 131 is semi-circular, and the shape of the first diversion part 133 is semi-circular ring-shaped arranged at the edge of the first liquid inlet part.

[0053] In some alternative embodiments, the thickness of the first diversion area 133 gradually decreases in the first direction, or the thickness of the first diversion area 133 remains unchanged in the first direction.

[0054] In the embodiments of the present application, the detection chip further includes a second fixing area 15. The second fixing area 15 is communicated with the side of the first fixing area 13 far from the sample inlet 11. A capture antibody for specifically capturing the target antigen is grafted in the second fixing area 15, and the second fixing area 15 is used to capture and bind the molecularly imprinted polymer combined with the target antigen based on the capture antibody. Specifically, a detection structure is arranged in the second fixing area 15, and the detection structure includes silica detection units arranged in an array, and the capture antibody is grafted on the surface of the silica detection units arranged in the array.

[0055] In some alternative embodiments, the second fixing area 15 includes: a second liquid inlet portion disposed on one side close to the sample inlet 11 and a second fixing portion on the side away from the sample inlet 11. The width of the second liquid inlet portion gradually increases in the first direction, and the first direction is the arrangement direction of the sample inlet 11 and the first fixing area 13; a second diversion portion disposed at the edge of the second liquid inlet portion for guiding the liquid to flow forward along the second diversion portion to the second liquid inlet portion. Wherein, the thickness of the second diversion portion is greater than the thickness of the second liquid inlet portion and the second fixing portion, so that the second diversion portion and the second liquid inlet portion form a stepped structure.

[0056] In some alternative embodiments, the shape of the second liquid inlet portion is semicircular, and the shape of the second diversion portion is a semi-circular ring disposed at the edge of the second liquid inlet portion.

[0057] In some alternative embodiments, the thickness of the second diversion area gradually decreases in the first direction, or the thickness of the second diversion area remains unchanged in the first direction.

[0058] In some alternative embodiments, the detection substrate further includes: a first mixing area 12 disposed between the sample inlet 11 and the first fixing area 13 for mixing the sample injected from the sample inlet 11 and then inputting it into the first fixing area 13; a second mixing area 14 disposed between the first fixing area 13 and the second fixing area 15 for mixing the molecularly imprinted polymer combined with the target antigen and then inputting it into the second fixing area 15. Optionally, in order to further improve the mixing effect of the first mixing area 12 and the second mixing area 14 on the liquid, the first mixing area 12 and the second mixing area 14 are serpentine microfluidic channels.

[0059] In some alternative embodiments, the detection substrate further includes: a waste liquid pool 16 communicated with the side of the second fixing area 15 away from the first fixing area 13 for collecting and caching the waste liquid after cleaning; the cover plate further includes: a sample outlet 17 communicated with the side of the waste liquid pool 16 away from the second fixing area 15, and the sample outlet 17 is used for discharging the waste liquid.

[0060] Figure 5 Shows a schematic diagram of the detection principle of a detection chip proposed in an embodiment of the present application for detecting a target antigen, as Figure 5As shown, the sample to be detected is injected from the sample inlet 11 of the detection chip, enters the first immobilization region 13 after being mixed in the first mixing region 12. The first antigen site 2 of the molecularly imprinted polymer and the second antigen site 5 on the fluorescent antibody 4 pre-set in the first immobilization region 13 bind to the target antigen 6 present in the sample, obtaining a molecularly imprinted polymer bound to the target antigen 6. The molecularly imprinted polymer bound to the target antigen 6 enters the second immobilization region 15 after being mixed in the second mixing region 14. The capture antibody 7 in the second immobilization region 15 reacts with and specifically binds to the target antigen 6 on the molecularly imprinted polymer bound to the target antigen 6, fixing the molecularly imprinted polymer with the fluorescent antibody 4 in the second immobilization region 15 through the target antigen 6, and completing the capture of the molecularly imprinted polymer bound to the target antigen 6. After the reaction is completed, a buffer solution (such as PBS buffer solution) is pumped into the sample inlet 11 of the detection chip to wash the second immobilization region 15 after the reaction, washing away the unfixed molecularly imprinted polymer, and the waste liquid flows into the waste liquid pool 16. Finally, the detection chip is placed under a fluorescence microscope for optical signal detection, observing the fluorescence signal generated by the fluorescent antibody 4 on the molecularly imprinted polymer fixed in the second immobilization region 15, so as to judge the content of the target antigen 6 in the sample.

[0061] In the embodiment of the present application, since multiple fluorescent antibodies 4 are carried on the molecularly imprinted polymer provided by the embodiment of the present application, each molecularly imprinted polymer fixed in the second immobilization region 15 can generate a fluorescence signal through the multiple fluorescent antibodies 4 thereon, thereby enhancing the fluorescence intensity in the second immobilization region 15 and effectively increasing the detection sensitivity.

[0062] In addition, when the existing antigen carrier is set in the first immobilization region 13, the freeze-drying method is usually used for setting, which makes the binding strength between the antigen carrier and the first immobilization region 13 relatively large. When the sample enters the first immobilization region 13, the re-dissolution effect of the freeze-dried antigen carrier is poor, thereby reducing the fluorescence intensity of the antigen carrier in the second immobilization region 15 subsequently. In the embodiment of the present application, since the molecularly imprinted polymer prepared by the preparation method provided by the embodiment of the present application is set in the first immobilization region 13, the molecularly imprinted polymer as an antigen carrier does not need to be set in a freeze-dried manner, thereby reducing the detachment difficulty between the molecularly imprinted polymer and the second immobilization region 13. After the sample enters the first immobilization region 13, the molecularly imprinted polymer can be more easily dissolved, improving the binding ability between the molecularly imprinted polymer and the target antigen, and further improving the fluorescence intensity of the molecularly imprinted polymer in the second immobilization region 15.

[0063] It is easy to understand that Figure 3Only an exemplary detection chip structure proposed in an embodiment of the present application is shown. Specifically, the specific shapes and structures of the first fixing area 13, the second fixing area 15, the first mixing area 12, the second mixing area 14, and the waste liquid pool 16 in the detection chip can be adjusted according to actual situations, and the present application does not limit this here.

[0064] Based on the same inventive concept, an embodiment of the present application proposes a method for preparing a molecularly imprinted polymer. Figure 6 A flowchart of a method for preparing a molecularly imprinted polymer is shown. As Figure 6 shown, the preparation method includes the following steps:

[0065] S101. Provide a substrate, and a plurality of first antigen sites and a plurality of antibody sites are arranged on the surface of the substrate.

[0066] When specifically implementing step S101, the substrate is prepared successively through the following steps:

[0067] S1011. Mix a first template molecule, a second template molecule, and a functional monomer to obtain a first solution. The first template molecule is the target antigen, and the second template molecule is the fluorescent antibody.

[0068] Mix a first template molecule, a second template molecule, and a functional monomer to obtain a first solution. Among them, the first template molecule is the target antigen, and the target antigen is the substance to be detected in the sample when the molecularly imprinted polymer is used for detection. Taking the target antigen as the first template molecule, during the formation of the three-dimensional network structure of the molecularly imprinted polymer, the first template molecule forms specific imprinting sites (i.e., first antigen sites) for the target antigen on the molecularly imprinted polymer, so that the molecularly imprinted polymer binds the target antigen in the sample through the specific imprinting sites for the target antigen during the detection of the sample. The second template molecule is the fluorescent antibody, and the fluorescent antibody is the substance that provides a fluorescent signal when the molecularly imprinted polymer is used for detection. Taking the fluorescent antibody as the second template molecule, the fluorescent antibody can specifically recognize the target antigen. During the formation of the three-dimensional network structure of the molecularly imprinted polymer, the second template molecule forms specific imprinting sites (i.e., antibody sites) for the fluorescent antibody on the molecularly imprinted polymer, so that the molecularly imprinted polymer binds the fluorescent antibody through the specific imprinting sites for the fluorescent antibody during the detection of the sample. The fluorescent antibody specifically binds the target antigen in the sample, and the fluorescent antibody in the molecularly imprinted polymer combined with the target antigen provides a fluorescent signal to realize the qualitative detection of the target antigen in the sample.

[0069] In some alternative embodiments, acrylamide is used as the functional monomer, the antigen solution of the target antigen is used as the first template molecule, and the Myo fluorescent antibody solution is used as the second template molecule. The first template molecule, the second template molecule, and the functional monomer are dissolved in PBS buffer for mixing to obtain the first solution. Among them, the pH of the PBS buffer is 6.4 - 7.4, and specific binding can be achieved between the fluorescent antibody of the second template molecule and the target antigen of the first template molecule.

[0070] In some alternative embodiments, the mass ratio of the first template molecule, the second template molecule, and the functional monomer in the first solution is 1:1:(3000 - 4000).

[0071] S1012: Add a crosslinking agent to the first solution to pre-polymerize the first template molecule, the second template molecule, and the functional monomer in the first solution to obtain a second solution.

[0072] The first solution contains the first template molecule, the second template molecule, and the functional monomer. In order to form a three-dimensional network structure of the molecularly imprinted polymer, it is necessary to first pre-polymerize the first template molecule, the second template molecule, and the functional monomer in the first solution so that the first template molecule, the second template molecule, and the functional monomer are connected by weak intermolecular forces.

[0073] In the embodiments of the present application, after obtaining the first solution, a crosslinking agent is added to the first solution to pre-polymerize the first template molecule, the second template molecule, and the functional monomer in the first solution to obtain a second solution. In the second solution, connections are established between the first template molecule, the second template molecule, the crosslinking agent, and the functional monomer through weak intermolecular forces such as hydrogen bonds. Specifically, the functional monomer and the crosslinking agent are used to form the three-dimensional network skeleton of the molecularly imprinted polymer, and the first template molecule and the second template molecule are used to generate specific spatial configurations and imprinting sites corresponding to the target antigen and the fluorescent antibody in the three-dimensional network skeleton formed by the functional monomer and the crosslinking agent. Therefore, the functional monomer and the crosslinking agent need to be connected in a way stronger than intermolecular forces such as hydrogen bonds, and the first template molecule and the second template molecule need to be detached from the three-dimensional network skeleton to form the specific spatial configurations (i.e., the first antigen site and the antibody site) that can bind the target antigen and the fluorescent antibody. Therefore, the first template molecule and the second template molecule respectively maintain the weak intermolecular forces with the functional monomer and the crosslinking agent.

[0074] In some alternative embodiments, the mass ratio of the functional monomer to the crosslinking agent is (3 to 4):1.

[0075] In some alternative embodiments, using N,N'-methylenebisacrylamide as the crosslinking agent, adding the crosslinking agent to the first solution, and after mixing evenly, placing it in a shaker at room temperature for pre-polymerization for 1 h - 2 h to form hydrogen bonds among the first template molecule, the second template molecule, the functional monomer, and the crosslinking agent, thereby obtaining the second solution.

[0076] S1013. Adding an initiator to the second solution to form covalent bonds between the functional monomer and the crosslinking agent in the second solution, thereby obtaining the first product.

[0077] In the embodiments of the present application, after obtaining the second solution, adding the initiator to the second solution, and under the action of the initiator, forming covalent bonds between the functional monomer and the crosslinking agent in the second solution to obtain the first product. In the first product, covalent bonds are formed between the functional monomer and the crosslinking agent to constitute the three-dimensional network skeleton of the molecularly imprinted polymer. The first template molecule and the second template molecule are connected to the three-dimensional network skeleton by intermolecular forces such as hydrogen bonds, and respectively occupy the first antigen site corresponding to the target antigen and the antibody site corresponding to the fluorescent antibody on the three-dimensional network skeleton.

[0078] In some alternative embodiments, the initiator includes a first initiator and a second initiator. After obtaining the second solution, using an ammonium persulfate solution with a mass fraction of 10% as the first initiator and tetramethylethylenediamine as the second initiator, performing nitrogen purging and deoxygenation treatment on the second solution for 10 min - 30 min, and sequentially adding the first initiator and the second initiator. After mixing evenly, reacting at room temperature for 24 h - 48 h to obtain the first product, wherein the volume fraction ratio of the first initiator to the second initiator is (10 to 2):1.

[0079] S1014. Removing the first template molecule and the second template molecule from the first product to form the plurality of first antigen sites and the plurality of antibody sites on the surface of the first product, thereby obtaining the matrix.

[0080] Since the first template molecule and the second template molecule in the first product occupy the first antigen site and the antibody site corresponding to the target antigen and the fluorescent antibody on the three-dimensional network skeleton, and the first template molecule and the second template molecule in the first product are connected to the functional monomer and the cross-linking agent through intermolecular forces respectively, therefore, by means of elution, the intermolecular forces between the first template molecule and the functional monomer and the cross-linking agent are destroyed, so that the first template molecule and the second template molecule are detached from the first product, forming the matrix.

[0081] After the first template molecule is detached from the first product, a first antigen site corresponding to the target antigen is formed on the three-dimensional network skeleton of the first product. The first antigen site is a specific cavity with a specific spatial configuration formed by detaching the first template molecule from the three-dimensional network skeleton. Since the specific spatial configuration of the first antigen site corresponds to the first template molecule, it can specifically bind to the corresponding target antigen. By means of elution, the intermolecular forces between the second template molecule and the functional monomer and the cross-linking agent are destroyed, so that the second template molecule is detached from the first product, and an antibody site corresponding to the fluorescent antibody is formed on the three-dimensional network skeleton of the first product. The antibody site is a specific cavity with a specific spatial configuration formed by detaching the second template molecule from the three-dimensional network skeleton. Since the specific spatial configuration of the antibody site corresponds to the second template molecule, it can specifically bind to the corresponding fluorescent antibody.

[0082] In some optional embodiments, a mixed solution of methanol and acetic acid is used as the eluent. The first product is washed multiple times with the eluent to elute the first template molecule and the second template molecule in the first product, and then dried, ground and sieved to obtain the matrix. Wherein, the washing time for each time is 1h - 2h, and the eluent is a mixed solution of methanol and acetic acid. Optionally, the number of times the eluent washes the first product is 3 times.

[0083] S102. Bind a plurality of fluorescent antibodies to the matrix. The fluorescent antibodies specifically bind to the matrix at the antibody site, and the fluorescent antibodies are used to generate fluorescent signals; wherein, a second antigen site is provided on each fluorescent antibody, and the first antigen site and the second antigen site are used to specifically bind to the target antigen.

[0084] In the specific implementation step S102, after obtaining the matrix, since the target antigen can be bound to the matrix through the first antigen site and the fluorescent antibody can be bound to the antibody site, in practical applications, after detecting the sample with the molecularly imprinted polymer, only the target antigen may exist in the sample. Therefore, it is necessary to pre-set the fluorescent antibody on the matrix so that the molecularly imprinted polymer combined with the fluorescent antibody can emit a fluorescent signal after capturing the target antigen in the sample, realizing the effective detection of the target antigen in the sample. Therefore, after obtaining the matrix, it is necessary to fill the antibody site in the matrix with the fluorescent antibody to obtain the molecularly imprinted polymer. In the molecularly imprinted polymer, the fluorescent antibody filled in the antibody site is connected to the matrix through intermolecular forces. Exemplarily, a hydrogen bond is formed between the fluorescent antibody filled in the antibody site and the matrix.

[0085] In an alternative embodiment, the matrix is placed in a dilution of the fluorescent antibody so that the fluorescent antibody fills the antibody site in the matrix, and the molecularly imprinted polymer is formed after vacuum drying. Each molecularly imprinted polymer contains multiple fluorescent antibodies corresponding to multiple antibody sites.

[0086] Based on the same inventive concept, the embodiments of the present application disclose an application of the molecularly imprinted polymer described in the embodiments of the present application in the detection of the target antigen in a sample.

[0087] Each embodiment in this specification is described in a progressive manner. The key points of each embodiment are the differences from other embodiments. The same or similar parts among the embodiments can be referred to each other.

[0088] As used herein, the terms "one embodiment", "an embodiment", or "one or more embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. In addition, please note that the examples of the phrase "in one embodiment" do not necessarily all refer to the same embodiment.

[0089] In the specification provided here, a large number of specific details are set forth. However, it can be understood that the embodiments of the present disclosure can be practiced without these specific details. In some instances, well-known methods, structures, and technologies have not been shown in detail so as not to obscure the understanding of this specification.

[0090] Although the preferred embodiments of the embodiments of the present application have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the embodiments of the present application.

[0091] Finally, it should also be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or terminal device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or terminal device. Without further limitation, an element defined by the statement "comprising an..." does not exclude the existence of additional identical elements in the process, method, article or terminal device comprising the said element.

[0092] The above has introduced in detail a molecularly imprinted polymer, its preparation method, application and detection chip provided by the present application. Specific examples are used in this article to elaborate on the principle and implementation manner of the present application. The description of the above embodiments is only used to help understand the method and its core idea of the present application; at the same time, for those of ordinary skill in the art, according to the idea of the present application, there will be changes in the specific implementation manner and application scope. In summary, the content of this specification should not be construed as a limitation to the present application.

Claims

1. A molecularly imprinted polymer, characterized in that, The molecularly imprinted polymer includes: a matrix, on the surface of which a plurality of first antigen sites and a plurality of antibody sites are provided; a plurality of fluorescent antibodies, which specifically bind to the matrix at the antibody sites and are used to generate fluorescence signals; wherein, each of the fluorescent antibodies is provided with a second antigen site, and the first antigen site and the second antigen site are used to specifically bind to a target antigen.

2. The molecularly imprinted polymer according to claim 1, wherein The antibody site is a specific cavity corresponding to the fluorescent antibody on the surface of the matrix, and the fluorescent antibody forms a connection with the matrix through intermolecular forces at the antibody site.

3. The molecularly imprinted polymer according to claim 1, wherein The first antigen site is a specific cavity corresponding to the target antigen on the surface of the matrix.

4. A detection chip, characterized in that, The detection chip includes a detection substrate and a cover plate located on one side of the detection substrate; The cover plate includes a sample inlet for injecting a sample; The detection substrate includes a first fixing area, which is communicated with the sample inlet, and the molecularly imprinted polymer according to any one of claims 1 to 3 is pre-set in the first fixing area, and the first fixing area is used to specifically bind to the target antigen in the sample based on the molecularly imprinted polymer.

5. The detection chip according to claim 4, wherein The detection substrate further includes: a second fixing area, which is communicated with the side of the first fixing area away from the sample inlet, and a capture antibody is grafted in the second fixing area, and the second fixing area is used to capture the molecularly imprinted polymer that has bound the target antigen based on the capture antibody.

6. The detection chip according to claim 4, wherein The first fixing area includes: a first liquid inlet part arranged on the side close to the sample inlet and a first fixing part arranged on the side away from the sample inlet, the width of the first liquid inlet part gradually increases along a first direction, and the first direction is the arrangement direction of the sample inlet and the first fixing area; a first diversion part, which is arranged at the edge of the first liquid inlet part and is used to make the sample flow forward along the first diversion part to the first liquid inlet part, wherein the thickness of the first diversion part is greater than the thicknesses of the first liquid inlet part and the first fixing part.

7. The detection chip according to claim 6, wherein The shape of the first liquid inlet part is semicircular, and the shape of the first diversion part is a semi-circular ring arranged at the edge of the first liquid inlet part.

8. A preparation method of a molecularly imprinted polymer, characterized in that, The method includes: providing a matrix, on the surface of which a plurality of first antigen sites and a plurality of antibody sites are provided; binding a plurality of fluorescent antibodies to the matrix, the fluorescent antibodies specifically bind to the matrix at the antibody sites and are used to generate fluorescence signals; wherein, each of the fluorescent antibodies is provided with a second antigen site, and the first antigen site and the second antigen site are used to specifically bind to a target antigen.

9. The preparation method of the molecularly imprinted polymer according to claim 8, wherein, The step of providing the matrix includes: mixing a first template molecule, a second template molecule and a functional monomer to obtain a first solution, wherein the first template molecule is the target antigen and the second template molecule is the fluorescent antibody; A crosslinking agent is added to the first solution to pre-polymerize the first template molecule, the second template molecule, and the functional monomer in the first solution, obtaining a second solution; An initiator is added to the second solution to form covalent bonds between the functional monomer and the crosslinking agent in the second solution, obtaining the first product; The first template molecule and the second template molecule in the first product are removed to form the plurality of first antigen sites and the plurality of antibody sites on the surface of the first product, obtaining the matrix.

10. Use of a molecularly imprinted polymer according to any one of claims 1 to 3 in the detection of a target antigen in a sample.

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