Lateral flow immunochromatography test paper as well as preparation method and application thereof

Abstract

The invention particularly discloses lateral flow immunochromatography test paper as well as a preparation method and application thereof, and relates to the technical field of biological medicines. The invention provides lateral flow immunochromatography test paper as well as a preparation method and application thereof. The test strip is green, efficient, high in sensitivity, convenient to operate and low in cost, and combined detection of multiple HCC markers can be achieved within a short time.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a lateral flow immunochromatographic test strip, its preparation method, and its application. Background Technology

[0002] Hepatocellular carcinoma (HCC) is highly malignant and rapidly progresses, but its onset is insidious. At the time of clinical diagnosis, 70%–80% of patients are already in the middle or late stages and cannot undergo radical surgical treatment. If the cancer is detected early and treated according to standardized protocols, the 5-year survival rate can exceed 50%. Promoting early screening for HCC is of great significance for improving patient survival and prognosis. Abnormal changes in liver cancer-related biomarkers in patient tissues or body fluids occur even in precancerous lesions, often indicating the presence or progression of the tumor earlier than imaging findings. However, current immunoassay techniques used for biomarker detection in clinical practice require professional personnel in medical institutions, resulting in low screening accessibility and poor public compliance. Therefore, developing on-site, real-time detection technologies for HCC biomarkers is beneficial for early warning, screening, and diagnosis of liver cancer, and can extend early cancer screening to the community and families, expanding the coverage of cancer screening and early diagnosis and treatment, thereby improving the early diagnosis rate of liver cancer and patient survival rates. Summary of the Invention

[0003] (a) Technical problems to be solved Therefore, one of the main objectives of this invention is to provide a lateral flow immunochromatographic test strip, its preparation method, and its application. This test strip is green, efficient, highly sensitive, easy to operate, and low in cost, and can achieve the combined detection of multiple HCC biomarkers in a short time.

[0004] (II) Technical Solution To achieve the above objectives, the present invention provides a detection reagent comprising a metallopolydopamine framework probe loaded with alpha-fetoprotein antibody and a metallopolydopamine framework probe loaded with abnormal prothrombin antibody.

[0005] In one embodiment, the metal polydopamine framework probe comprises a zinc-based metal-organic framework, a cobalt-based metal-organic framework, and / or a zirconium-based metal-organic framework.

[0006] In one embodiment, the metal polydopamine framework probe is a zinc-based metal-organic framework.

[0007] Metal-organic frameworks (MOFs) are three-dimensional coordination networks formed by the modular self-assembly of metal nodes (ions or clusters) and organic linkers. Their highly tunable structures and diverse topologies offer unique advantages as templates and precursors in the synthesis of various functional materials. Their large specific surface area and tunable pore structure enable efficient loading of large numbers of signal molecules (such as enzymes and fluorescent dyes), laying the foundation for cascaded signal amplification and ultra-high sensitivity detection. MOF derivatives, especially porous or hollow nanostructures, have been widely applied in gas adsorption, biosensing, catalysis, and drug delivery. Meanwhile, polydopamine (PDA), synthesized from the self-oxidative polymerization of dopamine, exhibits mussel-inspired strong adhesion properties and excellent bioconjugation capabilities, showing great promise in materials science, biosensing, and nanomedicine. Its surface is rich in functional groups such as amines, catechols, and imines, enabling it to easily, efficiently, and stably couple functional molecules via Michael addition or Schiff base reactions without complex activation steps, effectively maintaining the activity of biomolecules and perfectly solving the problem of difficult MOF self-coupling with antibodies. Secondly, PDA itself is dark brown-black with extremely strong visible light absorption, producing a high-contrast colorimetric signal, making it ideal for naked-eye interpretation and avoiding dependence on expensive instruments compared to traditional gold or fluorescent labels. Furthermore, it exhibits good biocompatibility, low toxicity, and excellent stability, and is easy to synthesize and inexpensive.

[0008] In another aspect, the present invention provides a method for preparing the above-mentioned detection reagent, comprising: The metal polydopamine framework was mixed with alpha-fetoprotein antibody and then incubated to obtain a metal polydopamine framework probe loaded with alpha-fetoprotein antibody. The metal polydopamine framework was mixed with an abnormal prothrombin antibody and incubated to obtain a metal polydopamine framework probe loaded with an abnormal prothrombin antibody.

[0009] In one embodiment, the concentration of the metal polydopamine framework is 1-5 mg / mL, and the concentrations of alpha-fetoprotein antibody and abnormal prothrombin antibody are 1-3 mg / mL.

[0010] In one embodiment, the concentration of the metal polydopamine framework is 1 mg / mL, and the concentrations of the alpha-fetoprotein antibody and the abnormal prothrombin antibody are 1 mg / mL.

[0011] In one embodiment, the incubation time is 4 to 6 hours.

[0012] In one embodiment, the above preparation method further includes: adding bovine serum albumin for blocking after incubation.

[0013] In another aspect, the present invention also provides a detection reagent obtained by the above preparation method.

[0014] In another aspect, the present invention also provides a lateral flow immunochromatographic test strip, comprising the above-mentioned detection reagents and sample pad, conjugate pad, nitrocellulose membrane, polyvinyl chloride plate, and absorbent pad.

[0015] In another aspect, the present invention also provides a method for preparing the above-mentioned lateral flow immunochromatographic test strip, comprising: The sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad are sequentially attached to a polyvinyl chloride plate. The metal polydopamine framework probe loaded with alpha-fetoprotein antibody is fixed on the nitrocellulose membrane to form detection line 1, and the metal polydopamine framework probe of abnormal prothrombin antibody is fixed on the nitrocellulose membrane to form detection line 2.

[0016] In one embodiment, the sample pad comprises glass fiber, nonwoven fabric, and / or polyester film.

[0017] In one embodiment, the sample pad is a polyester film.

[0018] In one embodiment, the sample pad is soaked in a solution containing 3-5% sucrose, 0.05-2% Triton-100, 0.05-0.1 mol / L tris(hydroxymethyl)aminomethane hydrochloride (pH 8.0), and phosphate buffer (pH 7.4) for 2-4 hours, and then dried at 35-42°C for 1-3 hours.

[0019] In one embodiment, the bonding pad comprises a polyester film and / or glass fiber.

[0020] In one embodiment, the bonding pad is a polyester film.

[0021] In one embodiment, the conjugate pad is soaked in a phosphate buffer solution (pH 7.4) containing 3-5% sucrose, 5-20% bovine serum albumin, and 0.5-5% Tween-20 for 2 hours, and then dried at 35-42°C for 1-3 hours. The metal polydopamine framework probe prepared above is prepared into a spraying solution with a concentration of 0.1-1 mg / mL, and sprayed onto the treated conjugate pad using a "gold spraying" mode, and then dried at 35-42°C for 1-3 hours.

[0022] In one embodiment, the absorbent pad is absorbent paper.

[0023] In one embodiment, the fixing method is spraying.

[0024] In one embodiment, the concentration of the metallopolydopamine framework probe loaded with alpha-fetoprotein antibody is 0.8~1.2 mg / mL.

[0025] In one embodiment, the concentration of the metal polydopamine framework probe loaded with abnormal prothrombin antibody is 0.8~1.2 mg / mL.

[0026] In one embodiment, a second antibody is also immobilized on the nitrocellulose membrane.

[0027] In one embodiment, the second antibody is goat anti-mouse IgG.

[0028] In one embodiment, the concentration of the second antibody is 0.8~1.2 mg / mL.

[0029] In another aspect, the present invention also provides a lateral flow immunochromatographic test strip, which is obtained by the above preparation method.

[0030] In another aspect, the present invention also provides a detection kit comprising the above-described detection reagents and / or lateral flow immunochromatographic test strips.

[0031] In another aspect, the present invention also provides the application of the above-mentioned detection reagents, lateral flow immunochromatographic test strips and / or detection kits in the preparation of products for detecting liver cancer.

[0032] (III) Beneficial Effects This invention provides a lateral flow immunochromatographic test strip, its preparation method, and its application. Compared with the prior art, it has the following advantages: 1. The test strips are easy to assemble and use, and the label has a strong colorimetric signal, enabling accurate qualitative and quantitative detection with high sensitivity and specificity.

[0033] 2. It can complete the joint detection of multiple targets in a short time, which greatly saves detection costs.

[0034] 3. The lowest detectable concentrations are 5 ng / mL and 10 ng / mL, respectively. All detections can be completed within 15 minutes. The test results can be observed with the naked eye. It has high specificity and sensitivity to the target substances, is easy to use, and the detection process is simple, fast and safe. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 This is a schematic diagram of a lateral flow immunochromatographic test strip.

[0037] Figure 2 This is a transmission electron microscope (TEM) image of Zn-MPF.

[0038] Figure 3X-ray diffraction pattern of Zn-MPF; Figure 4 The images show solutions of ZIF-8 and Zn-MPF under sunlight.

[0039] Figure 5 The graph shows the change in zeta potential after Zn-MPF-modified antibody.

[0040] Figure 6 This is the result of the colorimetric detection.

[0041] Figure 7 This is a standard curve of alpha-fetoprotein (AFP) test results.

[0042] Figure 8 This is a standard curve of abnormal prothrombin test results.

[0043] Figure 9 This is a comparison chart showing the consistency of lateral flow immunochromatographic assay strips in quantifying alpha-fetoprotein.

[0044] Figure 10 This is a comparison chart showing the quantitative consistency of lateral flow immunochromatographic test strips for abnormal prothrombin.

[0045] Figure 11 This is a specificity analysis diagram for the lateral flow immunochromatographic test strip. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] Terms and Definitions As used herein, the term "antibody" refers to a polypeptide comprising canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen, such as alpha-fetoprotein. As is known in the art, naturally occurring intact antibodies are tetrameric reagents of about 150 kDa, comprising two identical heavy-chain polypeptides (each about 50 kDa) and two identical light-chain polypeptides (each about 25 kDa), which associate with each other to form a structure commonly referred to as a "Y-shape". Each heavy chain contains at least four domains (each about 110 amino acids long): an amino-terminal variable (VH) domain (located at the tip of the Y-shape), followed by three constant domains: CHI, CH2, and a carboxyl-terminal CH3 (located at the base of the Y-stem). A short region called a "switch" connects the heavy-chain variable and constant regions. A "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region link the two heavy-chain polypeptides together in the intact antibody. Each light chain contains two domains: an amino-terminal variable (VL) domain followed by a carboxyl-terminal constant (CL) domain, separated from each other by another "switch". Those skilled in the art are familiar with the structural and sequence elements of antibodies, can identify the "variable" and "constant" regions in the provided sequence, and understand that there may be some flexibility in defining the "boundaries" between these domains, thereby allowing different representations of the same antibody chain sequence to, for example, indicate that such boundaries are shifted by one or more residues relative to different representations of the same antibody chain sequence.

[0048] A complete antibody tetramer consists of two heavy-chain-light-chain dimers, where the heavy and light chains are linked together by a single disulfide bond; two additional disulfide bonds link the heavy-chain hinge regions together, thus linking the dimers together to form a tetramer. Naturally occurring antibodies are also glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure characterized by an "immunoglobulin fold," formed by two β-sheets (e.g., 3, 4, or 5 folds) stacked on top of each other in compressed antiparallel β-barrels. Each variable domain contains three hypervariable loops called "complementarity-determining regions" (CDR1, CDR2, and CDR3) and four nearly invariant "framework" regions (FR1, FR2, FR3, and FR4). When the natural antibody folds, the FR regions form β-sheets to provide the structural framework of the domain, and the CDR loop regions from both the heavy and light chains aggregate in three-dimensional space, thus forming a single hypervariable antigen-binding site located at the tip of the Y-shaped structure. Naturally occurring antibodies have Fc regions that bind to elements of the complement system and also to receptors on effector cells, including, for example, effector cells that mediate cytotoxicity. As is known in the art, the affinity of the Fc region for Fc receptors and / or other binding properties can be modulated by glycosylation or other modifications. In some embodiments, antibodies generated and / or utilized according to the invention comprise glycosylated Fc domains, including modified or engineered such glycosylated Fc domains.

[0049] For the purposes of this invention, in some embodiments, any polypeptide or polypeptide complex including sufficient immunoglobulin domain sequences found in natural antibodies may be referred to as and / or used as an "antibody," regardless of whether the polypeptide is naturally occurring (e.g., produced by an organism reacting with an antigen) or produced by recombinant engineering, chemical synthesis, or other artificial systems or methods. In some embodiments, the antibody is polyclonal; in some embodiments, the antibody is monoclonal. In some embodiments, the antibody has constant region sequences that are specific to mouse, rabbit, primate, or human antibodies. In some embodiments, as known in the art, the antibody sequence elements are humanized, primate-derived, chimeric, etc.

[0050] Furthermore, the term "antibody" as used herein may, in appropriate embodiments, refer to (unless otherwise stated or clearly apparent from the context) any construct or form known or developed in the art for utilizing the structural and functional characteristics of an antibody in alternative representations. For example, in some embodiments, the antibodies used according to the invention have a form selected from, but not limited to, the following: intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies; antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or collections thereof; single-chain Fv; peptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies, such as IgNAR or fragments thereof); camel antibodies; masking antibodies; small modular immunotherapies; single-chain or tandem bifunctional antibodies; VHH; microantibodies; ankyrin repeats or DART; TCR-like antibodies. In some embodiments, the antibody may lack the covalent modifications (e.g., attachment glycans) that it would have in naturally occurring cases. In some embodiments, the antibody may contain covalent modifications (e.g., attached polysaccharides, effective loads (e.g., detectable portions, therapeutic portions, catalytic portions, etc.) or other side groups (e.g., polyethylene glycol, etc.).

[0051] Antibodies include antibody fragments. Antibodies also include, but are not limited to, polyclonal, monoclonal, chimeric dAb (domain antibody), single-chain, Fab, Fa, F(ab)2 fragments, scFv, and Fab expression libraries. Antibodies can be whole antibodies, immunoglobulins, or antibody fragments.

[0052] As used herein, the term "specific binding" means that an antibody or peptide can preferably bind to a binding partner, such as CD16A, in a competitive binding assay, as assessed using a recombinant form of the protein, an epitope therein, or a native protein present on the surface of isolated target cells. Competitive binding assays and other methods for determining specific binding are further described below and are well known in the art.

[0053] As used herein, the term "sample to be tested" refers to a composition obtained from or derived from a target subject that contains cellular entities and / or other molecular entities to be characterized and / or identified, for example, based on physical, biochemical, chemical, and / or physiological characteristics. This sample may be obtained from the subject's blood and other fluid and tissue samples of biological origin, such as biopsy tissue samples or tissue cultures or cells derived therefrom. The source of tissue samples may be solid tissue, such as fresh, frozen, and / or preserved organ or tissue samples, biopsy tissue or aspirates; blood or any blood component; body fluids; cells from any stage of an individual's pregnancy or development; or plasma.

[0054] In one embodiment, the sample to be tested includes, but is not limited to: tissue, blood, serum, plasma, blood-derived cells, lymph, synovial fluid, cerebrospinal fluid, pleural fluid, peritoneal fluid, bladder irrigation fluid, secretions (e.g., breast secretions), oral irrigation fluid, swabs (e.g., oral swabs), touch preparations, fine needle aspiration materials, cell extracts, and combinations thereof.

[0055] The detection reagents include those for detecting the biomarkers described herein by flow cytometry, Western blotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, near-infrared spectroscopy, immunochemiluminescence, capillary gel electrophoresis, or colloidal gold immunoassay.

[0056] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0057] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0058] Example 1: Preparation of raw materials for lateral flow immunochromatographic test strips: 1: Preparation of zinc-based metal-organic framework zeolite imidazole ester framework structure material-8 (ZIF-8): First, using 1~5 mmol zinc nitrate and 0.01~0.05 mmol 2-methylimidazole as precursors and 0.001~0.01 mmol hexadecyltrimethylammonium bromide as surfactant, monodisperse ZIF-8 was prepared by aqueous phase synthesis.

[0059] 2. Preparation of a zinc-based metal polydopamine framework (Zn-MPF) based on ZIF-8: Dissolve 10-20 mg of dopamine hydrochloride powder in 1-2 mL of ZIF-8 solution, then add pure water to bring the final concentration of dopamine in the reaction system to 1-2 mg / mL. Sonicate to form a homogeneous solution, then stir at 500 rpm for 1-2 h. Add 20-30 mL of Tris-HCl buffer (pH 8.5), and continue the reaction for 3-4 h. Afterward, collect the product by centrifugation, wash twice with pure water, and then disperse in pure water for storage.

[0060] like Figure 2 The image shown is a transmission electron microscope (TEM) image of Zn-MPF. The prepared Zn-MPF is cubic in shape with an average particle size of approximately 138.25 ± 11.52 nm.

[0061] like Figure 3The image shows the X-ray diffraction pattern of Zn-MPF. Zn-MPF retains clear characteristic peaks and highly matches the standard diffraction pattern of ZIF-8, corresponding to the non-centrosymmetric cubic crystal system, space group I-43m.

[0062] like Figure 4 The image shows the solutions of ZIF-8 and Zn-MPF under sunlight. ZIF-8 is a white solution with an opalescent sheen, while Zn-MPF is a dark black solution.

[0063] 3: Preparation of Zn-MPF probes (metal polydopamine framework probes): Take 1 mg / mL of Zn-MPF solution and incubate it with 1 mg / mL of monoclonal antibodies that recognize alpha-fetoprotein and abnormal prothrombin at room temperature for 4 h, respectively. Then add 10% bovine serum albumin for blocking, and you will get Zn-MPF probes loaded with alpha-fetoprotein antibody and Zn-MPF probes loaded with abnormal prothrombin antibody, respectively.

[0064] like Figure 5 As shown, the surface potential diagram of the Zn-MPF labeled detection probe (Zn-MPF-mAb) is obtained. As polydopamine is modified on the ZIF-8 surface and detection antibody is modified on the Zn-MPF surface to obtain Zn-MPF-mAb, the surface potential gradually changes from 28.77mV to -14.2mV to -9.18mV.

[0065] 4. Preparation of sample pads: Cut polyester film into sample pads 15-25 mm wide, and soak them in a solution containing 3-5% sucrose, 0.05-2% Triton-100, 0.05-0.1 mol / L tris(hydroxymethyl)aminomethane hydrochloride (pH 8.0), and phosphate buffer (pH 7.4) for 2-4 hours, and then dry them at 35-42℃ for 1-3 hours.

[0066] 5. Preparation of conjugate pads: The polyester film was cut into conjugate pads with a width of 10-20 mm and treated with a phosphate buffer solution (pH 7.4) containing 3-5% sucrose, 5-20% bovine serum albumin and 0.5-5% Tween-20, and dried at 35-42℃ for 1-3 h; the prepared metal polydopamine framework-labeled detection probe was prepared into a spraying solution with a concentration of 0.1-1 mg / mL and sprayed onto the treated conjugate pads using the "gold spraying" mode, and dried at 35-42℃ for 1-3 h.

[0067] 6: Preparation of absorbent pads: Cut absorbent paper into absorbent pads with a width of 15~25mm.

[0068] 7. Nitrocellulose membrane treatment: Prepare a target monoclonal capture antibody spray solution with a concentration of 0.8~1.2 mg / mL. Spray the solution onto the nitrocellulose membrane in multiple intervals using the "scratch" method to obtain two detection lines 3~5 mm apart. Starting from the direction of the sample pad, these lines are designated as detection line 1 and detection line 2. Prepare a goat anti-mouse IgG spray solution with a concentration of 0.8~1.2 mg / mL as a quality control line. Spray the solution onto the nitrocellulose membrane using the "scratch" method, controlling the distance between the control line and detection line 2 to be 3~5 mm and the distance between the control line and the end of the nitrocellulose membrane to be 5~10 mm.

[0069] Example 2: Preparation of lateral flow immunochromatographic test strips: Preparation of lateral flow immunochromatographic test strips: The nitrocellulose membrane prepared in Example 1 was adhered to a polyvinyl chloride (PVC) plate. Then, from one side of the PVC backing plate to the other, the sample pad, conjugate pad, and absorbent pad were sequentially adhered, with a 2-4 mm overlap between the sample pad and the conjugate pad, and between the conjugate pad and the absorbent pad and the nitrocellulose membrane. The assembled test strips were dried at 35-42℃ for 1-3 hours, and then stored at 4℃ in the dark for later use. Figure 1 : 1 Sample pad, 2 Binding pad, 3 Nitrocellulose membrane, 4 Detection line 1, 5 Detection line 2, 6 Quality control line, 7 Absorbent pad, 8 Polyvinyl chloride plate.

[0070] Example 3: Validation of the lateral flow immunochromatographic test strip: The sample to be tested is dropped onto the sample pad of the lateral flow immunochromatographic test strip prepared in Example 2. Under the action of capillary force, the sample solution migrates towards the nitrocellulose membrane. First, it recognizes and binds to the metal polydopamine framework-labeled detection probe coated on the conjugate pad, forming an immune complex of detection probe and target (alpha-fetoprotein, abnormal prothrombin) and is released from the conjugate pad. It further undergoes specific antigen-antibody recognition with the target monoclonal antibodies on detection lines 1 and 2, thereby forming a sandwich immune complex on the detection lines, causing the metal polydopamine framework-labeled detection probe to aggregate on detection lines 1 and 2. After a period of time, qualitative judgment is made directly by naked-eye observation. The RGB values ​​of the detection lines are analyzed using Color Picker software, and the concentration of the target in the sample is quantified according to the standard curve.

[0071] 1. Standard Curve Development: Standard antigen solutions of 0, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, and 500 ng / mL were prepared by serial dilution with 1× PBS buffer. 60 μL of each concentration of standard solution was sequentially added to the sample application area of ​​the test strip, and the reaction was allowed to proceed for 10 min. The signal intensity of the detection lines (T1 and T2 lines) was measured using the Color Picker software on a smartphone. A quantitative standard curve was established with the logarithm of the antigen concentration as the x-axis and the corresponding T-line signal intensity as the y-axis.

[0072] like Figure 7 The figure shows the standard curve for the detection of alpha-fetoprotein (AFP) using this immunochromatographic test strip platform, indicating that the quantitative linear range for AFP detection by this platform is 5–500 ng / mL. 2 =0.9924, the linear range for quantitative detection of abnormal prothrombin is 10−500ng / mL, R 2 =0.9921.

[0073] like Figure 8 The figure shows the standard curve for detecting abnormal prothrombin using this immunochromatographic test strip platform, indicating that the quantitative linear range for abnormal prothrombin detection by this platform is 10–500 ng / mL. 2 =0.9921.

[0074] 2. Determination of detection limit: The visual detection limit of the test strip is determined by visually interpreting the color intensity of the test line (T line) and comparing it with the negative control group.

[0075] like Figure 6 The image shows the colorimetric detection results of alpha-fetoprotein (AFP) and abnormal prothrombin using a Zn-MPF-based immunochromatographic test strip. For AFP samples with a concentration of ≥5 ng / mL, the test strip shows a clear color line after detection, with a detection limit of 5 ng / mL; for AFP samples with a concentration less than 5 ng / mL, the test strip does not show a color line. For abnormal prothrombin samples with a concentration of ≥10 ng / mL, the test strip shows a clear color line after detection, with a detection limit of 10 ng / mL; for abnormal prothrombin samples with a concentration less than 10 ng / mL, the test strip does not show a color line.

[0076] Example 4: Quantitative Consistency Test The quantitative results of the test strip and the clinical immunoassay were linearly fitted. The closer the Pearson correlation coefficient was to 1, the higher the quantitative consistency between the two methods.

[0077] like Figure 9As shown, the consistency of alpha-fetoprotein quantification between the test strip and the clinical immunoassay method is 0.9996, indicating that the detection platform is consistent with the clinical method in alpha-fetoprotein quantification.

[0078] like Figure 10 As shown, the quantitative consistency of the test strip with the clinical immunoassay method for abnormal prothrombin is 0.9777, indicating that the detection platform is consistent with the clinical method in the quantitative detection of abnormal prothrombin.

[0079] Example 5 Specificity test: The test strips are used to detect alpha-fetoprotein, abnormal prothrombin, and other interfering proteins such as carcinoembryonic antigen (CEA), pepsinogen I (PG I), pepsinogen II (PG II), neuron-specific enolase (NSE), human serum albumin (HSA), and bovine serum albumin (BSA). The color development of the test line is observed. If the test strips only produce a signal for the target antigens alpha-fetoprotein and abnormal prothrombin, it indicates good specificity.

[0080] like Figure 11 As shown, the test strip has specificity for detecting alpha-fetoprotein and abnormal prothrombin. The results indicate that the detection platform only generates a signal response for alpha-fetoprotein and abnormal prothrombin.

[0081] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0082] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A detection reagent, characterized in that, This includes metallopolydopamine framework probes loaded with alpha-fetoprotein antibodies and metallopolydopamine framework probes loaded with abnormal prothrombin antibodies.

2. The detection reagent according to claim 1, characterized in that, The metal polydopamine framework probes include zinc-based metal-organic frameworks, cobalt-based metal-organic frameworks, and / or zirconium-based metal-organic frameworks.

3. A method for preparing the detection reagent as described in claim 1, characterized in that, include: The metal polydopamine framework was mixed with alpha-fetoprotein antibody and then incubated to obtain a metal polydopamine framework probe loaded with alpha-fetoprotein antibody. The metal polydopamine framework was mixed with an abnormal prothrombin antibody and incubated to obtain a metal polydopamine framework probe loaded with an abnormal prothrombin antibody.

4. The preparation method according to claim 3, characterized in that, The concentration of the metal polydopamine framework is 1-5 mg / mL, and the concentrations of alpha-fetoprotein antibody and abnormal prothrombin antibody are 1-3 mg / mL.

5. A detection reagent, characterized in that, Obtained by the preparation method described in claim 3 or 4.

6. A lateral flow immunochromatographic test strip, characterized in that, Includes the detection reagents and sample pads, conjugation pads, nitrocellulose membranes, polyvinyl chloride plates, and absorbent pads as described in claims 1, 2, or 5.

7. A method for preparing the lateral flow immunochromatographic test strip as described in claim 6, characterized in that, include: The sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad are sequentially attached to a polyvinyl chloride plate. The metal polydopamine framework probe loaded with alpha-fetoprotein antibody is fixed on the nitrocellulose membrane to form detection line 1, and the metal polydopamine framework probe of abnormal prothrombin antibody is fixed on the nitrocellulose membrane to form detection line 2.

8. A lateral flow immunochromatographic test strip, characterized in that, It is obtained by the preparation method described in claim 7.

9. A test kit, characterized in that, Includes the detection reagents of claim 1, 2 or 5 and / or the lateral flow immunochromatographic test strips of claim 6 or 8.

10. The use of the detection reagent of claim 1, 2 or 5, the lateral flow immunochromatographic test strip of claim 6 or 8 and / or the detection kit of claim 9 in the preparation of products for detecting liver cancer.

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