Lateral flow immunochromatography kit based on metal ion chemical coordination signal amplification and preparation method thereof

Abstract

The invention relates to the technical field of immunodetection, in particular to a lateral flow immunochromatography kit based on metal ion chemical coordination signal amplification and a preparation method of the lateral flow immunochromatography kit. Aiming at the problem of insufficient lateral flow immunochromatography sensitivity at present, the invention develops an LFIA in-situ signal amplification strategy based on an in-situ coordination reaction of metal-based nanoparticles and a metal ion chromogenic reagent, and provides two lateral flow immunochromatography kits based on metal ion chemical coordination and a preparation method thereof. The kit provided by the invention is convenient to use, simple to operate, low in material performance requirement and low in cost, and can realize rapid and high-sensitivity detection of target detection substances such as disease markers, pathogenic microorganisms and small molecules to be detected. And background interference and irregular diffusion do not exist.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and in particular to a lateral flow immunochromatography kit based on metal ion chemical coordination signal amplification and a preparation method thereof. Background Art

[0002] Lateral flow immunochromatography (LFIA) is a rapid detection method that combines chromatography with immunoassay. It has the advantages of high stability, rapidity, portability, simplicity and user-friendliness. It has been widely used for rapid detection of target analytes and is currently the most commonly used point-of-care testing (POCT) platform. During the immunochromatography process, the signal probes gather on the T and C lines to form a colorimetric signal visible to the naked eye. Colloidal gold is the most commonly used LFIA detection probe due to its simple synthesis, stable properties and easy modification. LFIA based on colloidal gold is currently also widely used in disease diagnosis, environmental monitoring and food quality testing and other fields. However, due to the insufficient colorimetric intensity of colloidal gold and its low sensitivity in trace detection, it is currently only suitable for qualitative and semi-quantitative analysis of target objects, and it is difficult to meet the needs of clinical testing.

[0003] The signal amplification strategy based on LFIA is an important way to improve the sensitivity of LFIA detection. Usually, a high signal intensity detection probe or a special signal amplification strategy is used to react in situ at the detection line (T line), and a signal is generated at the T line by in situ growth of metal or by using enzyme-catalyzed substrate color to improve the sensitivity of detection (see references 1 and 2 for details). However, the enhancer used for metal growth often self-nucleates when reaching the T line, causing serious background interference (see reference 3 for details), and the enzyme-catalyzed substrate (such as TMB) often diffuses irregularly during the reaction, which has great limitations during use (see reference 4 for details).

[0004] Therefore, it is necessary to develop an in situ signal amplification method with controllable enhancement process and no background interference.

[0005] References 1-4, as follows:

[0006] 1.Khoris,IM;Takemura,K.;Lee,J.;Hara,T.;Abe,F.;Suzuki,T.;Park,EY,Enhanced colorimetric detection of norovirus using in-situ growth ofAg shellon Au NPs.Biosensors&Bioelectronics 2019,126,425-432.

[0007] 2.Chen, R.; Chen, X.; Zhou, Y.; Lin, T.; Leng, Y.; Huang, X.;

[0008] 3.Chu,

[0009] 4. Yao, N.; Li, Summary of the invention

[0010] The present invention aims at the problem of insufficient sensitivity of the current lateral flow immunochromatography, develops an LFIA in-situ signal amplification strategy based on the in-situ coordination reaction of metal-based nanoparticles and metal ion colorimetric reagents, and provides a lateral flow immunochromatography kit based on metal ion chemical coordination and a preparation method thereof. The kit of the present invention is easy to use, simple to operate, has low requirements on material performance and is low in cost, and can achieve rapid and highly sensitive detection of target detection objects such as disease markers, pathogenic microorganisms and small molecules to be detected. Moreover, there is no background interference and no irregular diffusion.

[0011] Metal ion coordination is a reaction that produces a strong color change by the coordination of metal ions with the corresponding color developer. Based on this, the inventors have developed a chemical coordination signal amplification technology through the metal ion clusters in the nanoparticles and the metal ion color developer. The metal ion signal amplification strategy is achieved by the metal nanoparticles fixed on the detection line and the color developer corresponding to the metal contained in them to form a colored complex through coordination. Taking zirconium-based, calcium-based and zinc-based nanoparticles (Zr-NPs, Ca-NPs, Zn-NPs) as an example, through Zr 4+ , Ca 2+ and Zn 2+ The corresponding ion colorimetric reagent undergoes a coordination reaction to generate a colored complex, and an in situ LFIA signal amplification strategy based on metal ion chemical coordination is developed to achieve in situ signal amplification of the T line.

[0012] The first aspect of the present invention provides a lateral flow immunochromatography kit based on metal ion chemical coordination signal amplification and a preparation method thereof. The lateral flow immunochromatography kit of the present invention can be used to detect disease markers, pathogenic microorganisms or small molecules to be detected.

[0013] The kit of the present invention comprises an independently arranged immunochromatographic test strip, a detection probe and a color developer;

[0014] The immunochromatographic test strip comprises a sample pad, a nitrocellulose membrane, an absorption pad and a backing substrate, wherein the sample pad, the nitrocellulose membrane and the absorption pad are sequentially pasted on the backing substrate and overlapped at adjacent locations, a detection line and a quality control line are arranged on the nitrocellulose membrane, the detection line is coated with an anti-disease marker monoclonal antibody 1, an anti-pathogenic microorganism monoclonal antibody 1 or a small molecule to be tested, and a secondary antibody is sprayed on the quality control line;

[0015] When the detection line is coated with anti-disease marker monoclonal antibody 1, the detection probe is a metal nanoparticle labeled with anti-disease marker monoclonal antibody 2, such as Zr-NPs, Ca-NPs or Zn-NPs metal nanoparticles;

[0016] When the detection line is coated with anti-pathogenic microorganism monoclonal antibody 1, the detection probe is a metal nanoparticle labeled with anti-pathogenic microorganism monoclonal antibody 2, such as Zn-NPs metal nanoparticles;

[0017] When the detection line is coated with a small molecule to be detected, the detection probe is a metal nanoparticle labeled with a monoclonal antibody against the small molecule to be detected, such as Zn-NPs metal nanoparticles.

[0018] Preferably, the disease marker is alpha-fetoprotein, the detection line is coated with anti-alpha-fetoprotein monoclonal antibody 1, and the detection probe is Zr-NPs or Ca-NPs labeled with anti-alpha-fetoprotein monoclonal antibody 2 (anti-AFP McAb2). The preparation method is as follows: take a Zr-NPs or Ca-NPs solution with a concentration of 100 μg / mL, and incubate it with anti-AFP McAb2 with a concentration of 1 mg / mL. The mass ratio of nanoparticles to monoclonal antibodies is 20:1, the incubation time is 1-2 hours, and then 1% bovine serum albumin is added to obtain labeled Zr-NPs or Ca-NPs and a detection probe with a concentration of 2 mg / mL.

[0019] Preferably, the disease marker is carcinoembryonic antigen, the detection line is coated with anti-carcinoembryonic antigen monoclonal antibody 1, and the detection probe is Zn-NPs labeled with anti-carcinoembryonic antigen monoclonal antibody 2 (anti-CEA McAb2). The preparation method is as follows: take a Zn-NPs solution with a concentration of 100 μg / mL, adjust the solution pH to 8-9, and incubate with anti-CEA McAb2 with a concentration of 1 mg / mL, the mass ratio of nanoparticles to monoclonal antibodies is 20:3, the incubation time is 4-6 hours, and then add 1% bovine serum albumin to obtain labeled Zn-NPs, and the concentration of the detection probe is 2 mg / mL.

[0020] Preferably, the pathogenic microorganism is Escherichia coli O157:H7; the detection line is coated with anti-Escherichia coli O157:H7 monoclonal antibody 1, and the detection probe is Zn-NPs labeled with anti-Escherichia coli O157:H7 monoclonal antibody 2 (anti-E. coli O157:H7 McAb2). The preparation method is as follows: take a Zn-NPs solution with a concentration of 100 μg / mL, adjust the solution pH to 8-9, and incubate with anti-E. coli O157:H7 McAb2 with a concentration of 1 mg / mL, the mass ratio of nanoparticles to monoclonal antibodies is 20:3, the incubation time is 4-6 hours, and then add 1% bovine serum albumin to obtain labeled Zn-NPs, and the concentration of the detection probe is 2 mg / mL.

[0021] Preferably, the small molecule to be detected is polymyxin B (PMB), the detection line is coated with polymyxin B, and the detection probe is Zn-NPs labeled with anti-polymyxin B monoclonal antibody (anti-PMB McAb). The preparation method is as follows: take a Zn-NPs solution with a concentration of 100 μg / mL, adjust the solution pH to 8-9, and incubate with anti-PMB McAb with a concentration of 1 mg / mL, the mass ratio of nanoparticles to monoclonal antibodies is 20:3, the incubation time is 4-6 hours, and then add 1% bovine serum albumin to obtain labeled Zn-NPs, and the concentration of the detection probe is 2 mg / mL.

[0022] Preferably, the secondary antibody is goat anti-mouse IgG.

[0023] Preferably, the method for preparing the immunochromatographic test strip comprises the following steps:

[0024] 1) Preparation and treatment of sample pad: cut the polyester fiber membrane into 20-25 mm width as the sample pad; soak it in sample pad treatment solution for 2-4 hours, and then bake it at 35-42°C for 1-3 hours;

[0025] 2) Treatment of nitrocellulose membrane: first, prepare the anti-disease marker monoclonal antibody 1, the anti-pathogenic microorganism monoclonal antibody 1 or the small molecule to be tested into a 1 mg / mL spray solution, spray it on the nitrocellulose membrane to obtain the test line; then prepare the secondary antibody into a spray solution with a concentration of 1 mg / mL, spray it on the nitrocellulose membrane to obtain the quality control line;

[0026] 3) Preparation of absorbent pad: Cut absorbent paper into 15-20 mm width as absorbent pad;

[0027] 4) Paste the nitrocellulose membrane in step 2) onto a polyvinyl chloride backing substrate, and then paste the sample pad and the absorption pad in sequence from one side to the other side of the polyvinyl chloride backing substrate.

[0028] More preferably, in step 1), the sample pad treatment solution contains 3-5 wt% sucrose, 0.05-2 wt% Triton-100, 0.05-0.1 mol / L Tris(OH)2Cl2 (pH 8.0), and 0.01 M phosphate buffered saline (pH 7.4).

[0029] More preferably, in step 2), the distance between the quality control line and the detection line is 3-5 mm, and the distance between the control line and the end of the nitrocellulose membrane close to the absorption pad is 5-10 mm.

[0030] More preferably, in step 4), the sample pad and the absorption pad overlap the nitrocellulose membrane by 2-4 mm respectively.

[0031] Preferably, the developer is a developer for Zr-NPs, a developer for Ca-NPs or a developer for Zn-NPs.

[0032] More preferably,

[0033] The method for preparing the color developer for Zr-NPs is as follows: adding Zr ion color developer arsenazo into a 3M hydrochloric acid solution; the mass volume ratio of arsenazo to the hydrochloric acid solution is 10 μg:200 μL;

[0034] The method for preparing the color developer for Ca-NPs is as follows: adding the Ca ion color developer arsenazo into a 0.01M phosphate buffer solution with a pH of 7.4; the mass volume ratio of arsenazo to the phosphate buffer solution is 10 μg:200 μL;

[0035] The method for preparing the color developing agent for Zn-NPs is as follows: adding a zinc color developing agent into a borate buffer solution with a pH of 8 to 9; the mass volume ratio of the zinc color developing agent to the borate buffer solution is 10 μg:200 μL.

[0036] The detection probes and test strips in this kit need to be stored in a 4°C refrigerator, and the color developer should be stored at room temperature.

[0037] The method for detecting a detection marker using the immunochromatographic kit comprises the following steps:

[0038] (1) Take the detection probe, add it to the sample to be tested, mix it evenly, and let it stand to obtain an immune complex formed by the target and the detection probe; the concentration of the detection probe is 1-2 mg / mL, and the immune complex formation time is 10-15 min.

[0039] (2) The immune complex obtained in step (1) is dripped onto the sample pad of the test strip. The sample solution moves toward the absorption pad under capillary action, and the detection probe combines with the target to form an immune complex. For disease markers and pathogenic microorganisms, the immune complex and the detection antibody on the detection line undergo specific antigen-antibody recognition to form an antibody-antigen-antibody sandwich structure, and the NPs are aggregated on the detection line; for small molecules to be tested, the detection probe that is not bound to the small molecules to be tested in the sample solution will undergo specific antigen-antibody recognition with the small molecules to be tested on the detection line, and the NPs are aggregated on the detection line. The excess probes are aggregated on the quality control line. The colorimetric reading uses software to read the R value of the RGB of the test strip T line. After the reading is completed, 20 μL of a color developer is dripped onto the T line in situ as a signal enhancement solution, and the software is used to read the R value of the RGB of the test strip T line to achieve quantitative judgment of the concentration of the target in the sample to be tested.

[0040] The detection principle of the above-mentioned immunochromatographic kit is as follows: when using the kit for detection, the sample to be tested is first incubated with the detection probe, and the target is combined with the detection probe to form an immune complex. For disease markers and pathogenic microorganisms, when flowing through the detection line, the immune complex combines with the monoclonal antibody fixed on the detection line to form an antibody-antigen-antibody "double antibody sandwich" structure, and the detection probe is aggregated on the detection line to generate a colorimetric signal; for small molecules to be tested, when flowing through the detection line, the target to be tested in the sample will "compete" with the target fixed on the detection line to bind to the antibody on the probe, resulting in a positive signal or weak signal, and a negative strong signal. The excess probe is combined with the secondary antibody (sheep anti-mouse IgG) on the quality control line. After the colorimetric signal is read, a metal ion color developer is added in situ on the detection line as a signal enhancement solution. After waiting for five minutes, the enhanced colorimetric signal on the detection line is read.

[0041] In addition, the final product of the kit of the present invention can also pre-embed the detection probe of the metal-based nanoparticles modified with the detection target in the binding pad, and only needs to drop the sample to be detected when using it.

[0042] In the second aspect of the present invention, the present invention also provides another lateral flow immunochromatography kit based on metal ion chemical coordination and a preparation method thereof.

[0043] The second lateral flow immunochromatography kit based on metal ion chemical coordination of the present invention can also be used to detect disease markers, pathogenic microorganisms or small molecules to be detected, and the kit includes independently set immunochromatography test strips and color developing agents;

[0044] The immunochromatographic test strip comprises a sample pad, a binding pad embedded with a detection probe, a nitrocellulose membrane, an absorption pad and a backing substrate. The sample pad, the binding pad embedded with the detection probe, the nitrocellulose membrane and the absorption pad are sequentially pasted on the backing substrate and overlapped at adjacent locations. A detection line and a quality control line are arranged on the nitrocellulose membrane. The detection line is coated with an anti-disease marker monoclonal antibody 1, an anti-pathogenic microorganism monoclonal antibody 1 or a small molecule to be detected, and a secondary antibody is sprayed on the quality control line.

[0045] When the detection line is coated with anti-disease marker monoclonal antibody 1, the detection probe is a metal nanoparticle labeled with anti-disease marker monoclonal antibody 2, such as Zr-NPs, Ca-NPs or Zn-NPs metal nanoparticles;

[0046] When the detection line is coated with anti-pathogenic microorganism monoclonal antibody 1, the detection probe is a metal nanoparticle labeled with anti-pathogenic microorganism monoclonal antibody 2, such as Zn-NPs metal nanoparticles;

[0047] When the detection line is coated with a small molecule to be detected, the detection probe is a metal nanoparticle labeled with a monoclonal antibody against the small molecule to be detected, such as Zn-NPs metal nanoparticles.

[0048] Preferably, the anti-disease marker is alpha-fetoprotein, the detection line is coated with anti-alpha-fetoprotein monoclonal antibody 1, the detection probe is Zr-NPs or Ca-NPs labeled with anti-alpha-fetoprotein monoclonal antibody 2 (anti-AFP McAb2), and the preparation method of the detection probe is the same as that in the first kit.

[0049] Preferably, the anti-disease marker is carcinoembryonic antigen; the detection line is coated with anti-carcinoembryonic antigen monoclonal antibody 1, the detection probe is Zn-NPs labeled with anti-carcinoembryonic antigen monoclonal antibody 2 (anti-CEA McAb2), and the preparation method of the detection probe is the same as that in the first kit.

[0050] Preferably, the pathogenic microorganism is Escherichia coli O157:H7; the detection line is coated with anti-Escherichia coli O157:H7 monoclonal antibody 1, the detection probe is Zn-NPs labeled with anti-E. coli O157:H7 monoclonal antibody 2 (anti-E. coli O157:H7 McAb2), and the preparation method of the detection probe is the same as that in the first kit.

[0051] Preferably, the small molecule to be detected is polymyxin B (PMB), the detection line is coated with polymyxin B, the detection probe is Zn-NPs labeled with anti-polymyxin B monoclonal antibody (anti-PMB McAb), and the detection probe and preparation method are the same as those in the first kit.

[0052] Preferably, the secondary antibody is goat anti-mouse IgG.

[0053] In the second kit, the preparation method of the immunochromatographic test strip is the same as the first three steps of the preparation method of the immunochromatographic test strip of the first kit, and the steps after step 3) are as follows:

[0054] 4) Preparation of a conjugate pad for embedding detection probes: first, soak a 16 mm wide polyester film in a conjugate pad treatment solution for 1-2 h, dry it at 37° C., then spray the prepared detection probe on the conjugate pad at an amount of 5 μg / mm, and assemble it with a sample pad, a nitrocellulose membrane, and a water-absorbing pad to prepare a test paper;

[0055] 5) Paste the nitrocellulose membrane onto the polyvinyl chloride backing substrate, and then paste the absorption pad, conjugation pad and sample pad in sequence from one side of the polyvinyl chloride backing substrate to the other side, wherein the conjugation pad and the absorption pad overlap the nitrocellulose membrane by 3 mm respectively.

[0056] In the step 4), the binding pad treatment solution contains 5% sucrose, 5% BSA and 0.5% Tween-20.

[0057] In the second kit, the color developer and the preparation method of each color developer are the same as the preparation method in the technical solution of the first kit.

[0058] The method for detecting antigens using the second immunochromatographic kit includes the following steps: taking a sample to be tested and dropping it onto the sample pad of the test paper, the sample liquid moves to the absorption pad under capillary action, and when the sample liquid flows through the binding pad, it performs specific antigen-antibody recognition with the detection probe pre-embedded in the binding pad to form an immune complex. For disease markers and pathogenic microorganisms, the immune complex performs specific antigen-antibody recognition with the detection antibody on the detection line to form an antibody-antigen-antibody sandwich structure, and NPs are aggregated on the detection line; for small molecules to be tested, the detection probe that is not bound to the small molecules to be tested in the sample solution will perform specific antigen-antibody recognition with the small molecules to be tested on the detection line, and NPs are aggregated on the detection line. The redundant probes are aggregated on the quality control line. The colorimetric reading uses software to read the R value of the RGB of the test paper T line. After the reading is completed, 20 μL of a color developer is added to the T line in situ as a signal enhancement solution, and the software is used to read the R value of the RGB of the test paper T line to achieve quantitative judgment of the concentration of the target in the sample to be tested.

[0059] The detection principle of the second kit is as follows: when using the kit for detection, the sample to be tested is directly added to the sample pad. When flowing through the binding pad, it performs specific antigen-antibody recognition with the detection probe pre-embedded in the binding pad to form an immune complex. For disease markers and pathogenic microorganisms, when flowing through the detection line, the immune complex binds to the monoclonal antibody fixed on the detection line to form an antibody-antigen-antibody "double antibody sandwich" structure, and the detection probe is aggregated on the detection line to generate a colorimetric signal; for small molecules to be tested, when flowing through the detection line, the target to be tested in the sample will "compete" with the target fixed on the detection line for binding to the antibody on the probe, resulting in a positive signal or weak signal, and a negative signal. The excess probe binds to the secondary antibody (sheep anti-mouse IgG) on the quality control line. After the colorimetric signal is read, a metal ion color developer is added in situ on the detection line as a signal enhancement solution. After waiting for five minutes, the enhanced colorimetric signal on the detection line is read.

[0060] Compared with the prior art, the present invention has the following significant advantages:

[0061] (1) The chemical coordination signal amplification immunochromatographic test strip based on metal-based nanoparticles and metal ion color developing agents of the present invention is simple to assemble and easy to use, and can effectively improve the detection sensitivity of the test strip. The amplified signal will not cause diffusion, has no background interference, and is easy to read.

[0062] (2) The detection kit based on coordination chemistry of the present invention can realize the enhancement of visual output signal of colorimetry. The colorimetric detection limit of AFP after amplification is as low as 5 ng / mL, the colorimetric detection limit of CEA after amplification is 0.5 ng / mL, and the colorimetric detection limit of Escherichia coli O157:H7 is as low as 10 4 CFU / mL, the quantitative range of polymyxin B is expanded from 100-500ng / mL to 100-1000ng / mL. The test results are accurate and reliable, and can meet the needs of high-sensitivity detection of different target objects. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 It is a schematic diagram of the lateral flow chromatography test strip for disease markers and pathogenic microorganisms of the present invention; wherein 1 is a sample pad, 2 is a nitrocellulose membrane, 3 is a detection line (immobilized antibody), 4 is a quality control line, 5 is an absorption pad, and 6 is a polyvinyl chloride plate.

[0064] Figure 2 It is a schematic diagram of the lateral flow chromatography test strip for the small molecules to be detected of the present invention; wherein 1 is a sample pad, 2 is a nitrocellulose membrane, 3 is a detection line (fixed small molecules), 4 is a quality control line, 5 is an absorption pad, and 6 is a polyvinyl chloride plate.

[0065] Figure 3 middle, Figure 3 -a is a transmission electron microscope image of Zr-NPs of the present invention, Figure 3 -b is a transmission electron microscopy image of the Zn-NPs of the present invention.

[0066] Figure 4 On-site color conversion diagram of chemical coordination signal amplification of Zr-NPs, Ca-NPs, and Zn-NPs.

[0067] Figure 5 middle, Figure 5 -a is the UV spectrum verifying the principle of chemical coordination signal amplification of Zr-NPs, Figure 5 -b is the UV spectrum verifying the principle of chemical coordination signal amplification of Zn-NPs.

[0068] Figure 6 The colorimetric detection results and standard curve of Zr-NPs and AFP detection based on chemical coordination signal amplification immunochromatographic test strips are shown. Figure 6 -a is the result of the pre-enhancement test strip, with a detection limit of 25ng / mL. Figure 6 -b is the enhanced result with a detection limit of 5 ng / mL.

[0069] Figure 7 The colorimetric results and standard curve of Zn-NPs and the colorimetric detection of CEA based on the chemical coordination signal amplification immunochromatographic test strips are shown. Figure 7-a is the result of the pre-enhancement test strip, with a detection limit of 10ng / mL. Figure 7 -b is the enhanced result with a detection limit of 0.5 ng / mL.

[0070] Figure 8 The colorimetric results and standard curve of Zn-NPs and the immunochromatographic test strips based on chemical coordination signal amplification for the detection of E.coli O157:H7 are shown. Figure 8 -a is the result of the pre-enhancement test strip, with a detection limit of 10 6 CFU / mL, Figure 8 -b is the enhanced result, with a detection limit of 10 4 CFU / mL.

[0071] Fig. 9 The colorimetric detection results and standard curve of Zn-NPs and PMB detection based on chemical coordination signal amplification immunochromatographic test strips are shown. Fig. 9 -a is the result of the pre-enhancement test strip, and the detection quantitative range is 100-500ng / mL, Fig. 9 -b is the enhanced result, with a detection limit of 100-1000ng / mL.

[0072] Fig.10 To verify the diffusion-free in-situ deposition performance of the horseradish peroxidase catalytic reaction of the present invention, Fig.10 -a is the result of horseradish peroxidase catalyzed reaction, Fig.10 -b is the metal ion coordination result of the present invention.

[0073] Fig.11 To verify the background interference-free performance of the present invention with metal in-situ growth, Fig.11 -a is the result of metal in-situ growth, Fig.11 -b is the metal ion coordination result of the present invention.

[0074] Fig.12 It is a schematic diagram of the second lateral flow chromatography test strip of the present invention; wherein 1 is a sample pad, 2 is a binding pad of a Zn-NPs detection probe coated with modified CEA monoclonal antibody, 3 is a detection line, 4 is a quality control line, 5 is a nitrocellulose membrane, 6 is an absorption pad, and 7 is a polyvinyl chloride plate.

[0075] Fig.13 The color development result of the Zn-NPs colorimetric immunochromatographic test strip with a binding pad in the second detection kit of the present invention for detecting CEA, Fig.13 -a is the actual finished product display picture, Fig.13 -b is the test result of the test paper before enhancement, Fig.13 -c is the enhanced test paper test result. DETAILED DESCRIPTION

[0076] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0077] In the present invention, the materials involved are described as follows:

[0078] AFP and CEA antigens were purchased from Suzhou Jinan Protein Technology Co., Ltd.; Escherichia coli O157:H7 strains were purchased from Mingzhu Biotechnology, and polymyxin B was purchased from Bio-Aolong; AFP and CEA paired monoclonal antibodies were purchased from Shanghai Lingchao Biotechnology Co., Ltd.; Escherichia coli O157:H7 paired monoclonal antibodies and polymyxin B monoclonal antibodies were purchased from ThermoFisherSCIENTIFIC, and goat anti-mouse IgG was purchased from Jackson ImmunoResearch; sample pads, conjugate pads, and absorption pads were purchased from Hangzhou Fenghang Technology Co., Ltd.; nitrocellulose membrane (NC membrane) was purchased from Sartorius, Germany, and the raw materials for synthesizing metal nanoparticles and metal ion colorimetric reagents were purchased from Aladdin Reagents (Shanghai) Co., Ltd.;

[0079] Sample pad treatment solution: containing 5wt% sucrose, 1wt% Triton-100, 0.1M Tris(OH)2Cl2 (pH 8.0), and 0.01M phosphate buffered saline (pH 7.4);

[0080] Conjugate pad treatment: 5% sucrose, 5% BSA, 0.5% Tween-20, 0.01 M phosphate buffered saline (pH 7.4).

[0081] Preparation method of color developer for Zr-NPs: adding Zr ion color developer arsenazo into 3M hydrochloric acid solution; the mass volume ratio of arsenazo to hydrochloric acid solution is 10 μg:200 μL;

[0082] Preparation method of color developer for Ca-NPs: adding Ca ion color developer arsenazo into 0.01M phosphate buffer solution with pH 7.4; the mass volume ratio of arsenazo to phosphate buffer solution is 10 μg:200 μL;

[0083] Preparation method of color developing agent for Zn-NPs: Add zinc color developing agent into borate buffer with pH of 8 to 9. The mass volume ratio of zinc color developing agent to borate buffer is 10 μg:200 μL.

[0084] Example 1 Immunochromatographic Kit for Detecting Disease Marker Alpha-Fetoprotein AFP

[0085] 1.1 The immunochromatographic test kit for detecting the disease marker alpha-fetoprotein of this embodiment includes independently arranged immunochromatographic test strips, detection probes and color developing agents;

[0086] like Figure 1 As shown, the LFIA test strip includes a sample pad 1, a nitrocellulose membrane 2, an absorption pad 5 and a polyvinyl chloride backing substrate 6. The sample pad 1, the nitrocellulose membrane 2 and the absorption pad 5 are sequentially pasted on the polyvinyl chloride backing substrate 6, overlapping at adjacent locations, and the sample pad and the absorption pad overlap the nitrocellulose membrane by 3 mm, respectively. The nitrocellulose membrane 2 is provided with a detection line 3 and a quality control line 4, the detection line is sprayed with anti-alpha-fetoprotein monoclonal antibody 1 (anti-AFP McAb1), and the quality control line is sprayed with a secondary antibody (sheep anti-mouse IgG).

[0087] The detection probe is Zr-NPs labeled with anti-alpha-fetoprotein monoclonal antibody 2 (anti-AFP McAb2).

[0088] The color developing agent was 200 μL of 3M hydrochloric acid solution containing 10 μg of arsenazo (20 μL was added dropwise when used).

[0089] 1.2 The preparation method of the immunochromatographic test strip comprises the following steps:

[0090] 1) Preparation and treatment of sample pad: cut the polyester fiber membrane into 22 mm width as the sample pad; soak it in sample pad treatment solution for 2 h, and then bake it at 35-42 ° C for 2 h;

[0091] 2) Treatment of nitrocellulose membrane: first, prepare anti-alpha-fetoprotein monoclonal antibody 1 into a 1 mg / mL spray solution, and spray it on the nitrocellulose membrane at a rate of 1 μL / cm to obtain a test line; then prepare the secondary antibody (sheep anti-mouse IgG) into a 1 mg / mL spray solution, and spray it on the nitrocellulose membrane at a rate of 1 μL / cm to obtain a quality control line;

[0092] 3) Preparation of absorbent pad: Cut absorbent paper into 18 mm width to make absorbent pad;

[0093] 4) Paste the nitrocellulose membrane in step 2) onto a polyvinyl chloride backing substrate, and then paste the sample pad and the absorption pad in sequence from one side to the other side of the polyvinyl chloride backing substrate.

[0094] 1.3 The preparation method of the detection probe labeled with Zr-NPs comprises the following steps:

[0095] 1) Take 1 mg of anti-alpha-fetoprotein monoclonal antibody 2 (anti-AFP McAb2) and add 1 mL of phosphate buffered saline to prepare a 1 mg / mL anti-alpha-fetoprotein monoclonal antibody 2 (anti-AFP McAb2) solution for use.

[0096] 2) The Zr-NPs labeled AFP-recognizing detection probe was prepared according to the following method: 2 mL of 100 μg / mL Zr-NPs solution was added with 10 μg of anti-AFP monoclonal antibody in phosphate buffered saline, and the mixture was rotated and mixed at room temperature for 2 h. Then, 1% bovine serum albumin solution was added to block unreacted sites, and the mixture was centrifuged. The resulting precipitate was diluted with 1 mL of phosphate buffered saline solution containing 1% bovine serum albumin and stored at 4°C for later use.

[0097] 1.4 Detection method:

[0098] Take 60 μL of the AFP antigen sample diluted with pH 7.4 phosphate buffer, add 12.5 μL of the prepared Zr-NPs labeled detection probe, mix well, let stand for 15 minutes, and then drip the mixed solution onto the sample pad of the test strip. The colorimetric reading uses the software to read the R value of the RGB of the test strip T line. After the reading is completed, drip 20 μL of the signal enhancement solution containing the metal ion color developer on the T line in situ, and use the software to read the R value of the RGB of the test strip T line to achieve quantitative judgment of the AFP concentration in the sample to be tested. The detection results before and after enhancement are as follows Figure 6 -a, b, the recovery results of AFP detection are shown in Table 1.

[0099] Table 1. AFP spike test results AFP spike test results

[0100]

[0101] As can be seen from Table 1, the recovery rates of the Zr-NPs detection kit for AFP in Example 1 before and after AFP enhancement were between 94.06% and 113.12%, which has good accuracy.

[0102] Example 2 Immunochromatographic Kit for Detecting Disease Marker Alpha-Fetoprotein AFP

[0103] 2.1 The kit of this example is basically the same as 1.1 in Example 1, except that the detection probe is Ca-NPs labeled with anti-alpha-fetoprotein monoclonal antibody 2 (anti-AFP McAb2), and the color developer is a color developer for Ca-NPs.

[0104] 2.2 The preparation method of the immunochromatographic test strip is the same as 1.2 in Example 1.

[0105] 2.3 The preparation method of the Ca-NPs labeled detection probe is basically the same as that of 1.3 in Example 1, except that the Zr-NPs solution is replaced with Ca-NPs of the same concentration.

[0106] 2.4 Detection method: It is basically the same as 1.4 in Example 1, except that the detection probe labeled with Zr-NPs is replaced with the detection probe labeled with Ca-NPs, and the color developer is replaced with the color developer for Ca-NPs.

[0107] Example 3 Immunochromatographic Kit for Detecting Disease Marker Carcinoembryonic Antigen CEA

[0108] 3.1 The kit of this embodiment is basically the same as 1.1 in Example 1, except that the detection line is sprayed with anti-carcinoembryonic antigen monoclonal antibody 1, the detection probe is Zn-NPs labeled with anti-carcinoembryonic antigen monoclonal antibody 2, and the color developer is a color developer for Zn-NPs.

[0109] 3.2 The preparation method of the immunochromatographic test strip is basically the same as that in 1.2 of Example 1, except that the anti-carcinoembryonic antigen monoclonal antibody 1 is sprayed onto the nitrocellulose membrane to form the detection line.

[0110] 3.3 Preparation method of Zn-NPs labeled detection probe:

[0111] 1) Take 1 mg of anti-CEA McAb2 and add 1 mL of phosphate buffered saline to prepare a 1 mg / mL anti-CEA McAb2 solution for later use.

[0112] 2) The detection probe for recognizing CEA labeled with Zn-NPs was prepared according to the following method: 2 mL of 100 μg / mL Zn-NPs solution was taken, 30 μg of anti-CEA monoclonal antibody was added, the pH of the solution was adjusted to 8-9, and the mixture was rotated and mixed at room temperature for 4 h, and then 1% bovine serum albumin solution was added to block unreacted sites, centrifuged, and the resulting precipitate was diluted with 1 mL of phosphate buffered saline solution containing 1% bovine serum albumin and stored at 4°C for later use.

[0113] 3.4 Detection method: It is basically the same as 1.4 in Example 1, except that the detection probe labeled with Zr-NPs is replaced with the detection probe labeled with Zn-NPs prepared in Example 3.3, and the color developer is replaced with the color developer for Zn-NPs. The detection results before and after enhancement are as follows: Figure 7 -a, b, and the recovery results of spiked CEA are shown in Table 2.

[0114] Table 2. CEA spike test results CEA spike test results

[0115]

[0116] As can be seen from Table 2, the recovery rates of the Zn-NPs detection kit for CEA in Example 3 before and after the enhancement of CEA detection were between 93.00% and 118.20%, which has good accuracy.

[0117] Example 4 Immunochromatographic Kit for Detecting Pathogenic Microorganism Escherichia coli O157:H7

[0118] 4.1 The kit of this embodiment is basically the same as 1.1 in Example 1, except that the detection line is sprayed with anti-Escherichia coli O157:H7 monoclonal antibody 1, the detection probe is Zn-NPs labeled with anti-Escherichia coli O157:H7 monoclonal antibody 2, and the color developer is a color developer for Zn-NPs.

[0119] 4.2 The preparation method of the immunochromatographic test strip is basically the same as that in 1.2 of Example 1, except that the anti-Escherichia coli O157:H7 monoclonal antibody 1 is sprayed onto the nitrocellulose membrane to form the detection line.

[0120] 4.3 Preparation method of Zn-NPs labeled detection probe:

[0121] 1) Take 1 mg of anti-E. coli O157:H7 McAb2 and add 1 mL of phosphate buffered saline to prepare a 1 mg / mL anti-E. coli O157:H7 McAb2 solution for later use.

[0122] 2) The detection probe for recognizing E. coli O157:H7 labeled with Zn-NPs was prepared according to the following method: 2 mL of 100 μg / mL Zn-NPs solution was taken, 30 μg of anti-E. coli O157:H7 monoclonal antibody 2 was added, the pH of the solution was adjusted to 8-9, and the mixture was rotated and mixed at room temperature for 4 h. Then, 1% bovine serum albumin solution was added to block the unreacted sites, and the mixture was centrifuged. The resulting precipitate was diluted with 1 mL of phosphate buffered saline solution containing 1% bovine serum albumin and stored at 4°C for later use.

[0123] 4.4 Detection method: It is basically the same as 1.4 in Example 1, except that the detection probe labeled with Zr-NPs is replaced with the detection probe labeled with Zn-NPs prepared in Example 4.3, and the color developer is replaced with the color developer for Zn-NPs. The detection results before and after enhancement are as follows: Figure 8 -as shown in a and b.

[0124] Example 5 Immunochromatographic Kit for Detecting Small Molecule Polymyxin B

[0125] 5.1 The kit of this embodiment is basically the same as 1.1 in embodiment 1, except that the detection line is sprayed with polymyxin B and the test paper structure is as follows Figure 2 As shown, the detection probe is Zn-NPs labeled with anti-polymyxin B monoclonal antibody, and the color developer is a color developer for Zn-NPs.

[0126] 5.2 The preparation method of the immunochromatographic test strip is basically the same as that in 1.2 of Example 1, except that polymyxin B is sprayed onto the nitrocellulose membrane to form the detection line.

[0127] 5.3 Preparation method of Zn-NPs labeled detection probe:

[0128] 1) Take 1 mg of anti-PMB McAb and add 1 mL of phosphate buffered saline to prepare a 1 mg / mL anti-PMB McAb solution for later use.

[0129] 2) The detection probe for recognizing PMB labeled with Zn-NPs was prepared according to the following method: 2 mL of 100 μg / mL Zn-NPs solution was added with 30 μg of anti-PMB monoclonal antibody, the pH of the solution was adjusted to 8-9, and the mixture was rotated and mixed at room temperature for 4 h. Then, 1% bovine serum albumin solution was added to block unreacted sites, and the mixture was centrifuged. The resulting precipitate was diluted with 1 mL of phosphate buffered saline containing 1% bovine serum albumin and stored at 4°C for later use.

[0130] 5.4 Detection method: It is basically the same as 1.4 in Example 1, except that the detection probe labeled with Zr-NPs is replaced with the detection probe labeled with Zn-NPs prepared in Example 5.3, and the color developer is replaced with the color developer for Zn-NPs. The detection results before and after enhancement are as follows: Fig. 9 -as shown in a and b.

[0131] Comparative Example 1: Generating a signal on the T line by in-situ growth of metal

[0132] Taking the in-situ growth of silver nanoparticles based on colloidal gold as an example, 2% hydroquinone solution and 0.2% silver nitrate solution were respectively prepared with pH = 4.0, 0.01M citric acid-sodium citrate buffer, and the silver growth solution was prepared in a volume ratio of 1:1, and prepared on demand. After the colloidal gold test paper completes the detection, 100uL of silver growth solution is added to the sample pad, and the enhancement is completed after 5 minutes. The enhancement results of comparative example 1 and the metal ion coordination enhancement results of Zn-NPs labeled with anti-carcinoembryonic antigen monoclonal antibody 2 in Example 3 of the present invention are compared with the background effect of nitrocellulose membrane as shown in the figure below. Fig.11 shown.

[0133] Comparative Example 2: Using enzyme to catalyze substrate color to generate signal on T line

[0134] Taking the enzyme catalytic reaction with TMB as the catalytic substrate as an example, prepare a 100mM H2O2 solution and a 50mM TMB solution, respectively, take 20uL of H2O2 and TMB solution and add them to 200uL pH = 4.0 acetic acid-sodium acetate buffer to prepare the enhanced solution, which is prepared on the spot. After the nanozyme test paper completes the detection, add 4uL of the enhanced solution in situ on the T line, and the enhancement is completed within 1min. The results are as follows Fig.10 -a as shown.

[0135] The detection methods involved in Examples 1-5 and Comparative Examples 1-2 are as follows:

[0136] 1) The transmission electron microscopy images of Zr-NPs and Zn-NPs used in Example 1 and Example 3 were taken by transmission electron microscopy (FEI Tencnai T20). Figure 3 -a, Figure 3 -b as shown.

[0137] 2) The signal amplification effect detection method of the metal nanoparticles selected in Examples 1-5 is as follows: by reacting different masses (10 μg, 30 μg) of Zr, Ca and Zn NPs with 200 μL of a color developer solution, and photographing the color change of the solution under natural light with a mobile phone, the results are as follows Figure 4 The absorption spectrum at 300-800 nm was scanned by UV-3600+ spectrophotometer to observe whether the characteristic absorption peaks of colored coordination substances appeared at 665 nm and 620 nm. The results are shown in Figure 5 -as shown in a and b.

[0138] 3) The in-situ signal deposition performance verification method of the present invention is as follows: 1 mg / mL of anti-AFP monoclonal antibody 1 is fixed in the circular area of ​​the NC membrane, the Zr-NPs modified with anti-AFP monoclonal antibody 2 are incubated with 60 μL AFP antigen solution for 10 minutes, and the mixed solution is dripped into the circular area of ​​the NC membrane for reaction for 10 minutes. After 10 minutes, 30 μL of a developer solution containing azodicarbonyl is dripped to observe whether the range of the purple-red substance produced is within the circular area. The results are as follows Fig.10 -b as shown.

[0139] 4) The in-situ signal deposition performance verification method of Comparative Example 2 is as follows: 1 mg / mL anti-AFP monoclonal antibody 1 is fixed in the circular area of ​​the NC membrane, and 60 μL of 1 mg / mL horseradish peroxidase-modified secondary antibody (sheep anti-mouse IgG) is added to the circular area of ​​the NC membrane for reaction for 10 minutes. After 10 minutes, 30 μL of a color developer solution containing TMB and H2O2 is added to observe whether the range of blue substance generation is within the circular area. The results are shown in FIG. Fig.10 -a as shown.

[0140] The test results are described as follows:

[0141] like Figure 3 As shown, it is the transmission electron microscopy image of Zr-NPs and Zn-NPs used in Example 1 and Example 3. Figure 3 a shows that the synthesized Zr-NPs are uniformly dispersed irregular spherical particles with a particle size of 160-180 nm. Figure 3 b shows that Zn-NPs have a cubic structure, no obvious aggregation phenomenon, and the particle size is 100 nm.

[0142] like Figure 4 As shown, the Zr-NPs, Ca-NPs, and Zn-NPs used in Examples 1-5 all showed obvious color enhancement after reacting with the corresponding metal ion color developing reagents.

[0143] Figure 5 middle, Figure 5 a is the UV-Vis spectrum of Zr-NPs after coordination with arsenazo in Example 1. Arsenazo has a characteristic absorption peak at 525 nm. After the reaction of MOF and arsenazo, the resulting complex has an obvious absorption peak at 665 nm, proving that Zr-MOF and arsenazo are successfully coordinated. Figure 5 b is the characteristic absorption peak of the complex that also appears at 620 nm after the Zn-NPs in Example 3-6 are coordinated with the corresponding zinc reagent.

[0144] Figure 6 middle, Figure 6 a is the color development result of the Zr-NPs colorimetric immunochromatographic test strip in Example 1 for detecting AFP, and the detection limit is 25 ng / mL, Figure 6 b is the color development result of AFP detection by immunochromatographic test strip after chemical coordination signal amplification, with a detection limit of 5 ng / mL.

[0145] Figure 7 middle, Figure 7 a is the color development result of the Zn-NPs colorimetric immunochromatographic test strip in Example 3 for detecting CEA, with a detection limit of 10 ng / mL, Figure 7 b is the color development result of CEA detected by immunochromatographic test strip after chemical coordination signal amplification, with a detection limit of 0.5 ng / mL.

[0146] Figure 8 middle, Figure 8 a is the color development result of Zn-NPs colorimetric immunochromatographic test strip in Example 4 for detecting E.coli O157:H7, with a detection limit of 10 6 CFU / mL, Figure 8b is the color development result of immunochromatographic test strips for detecting E.coliO157:H7 after chemical coordination signal amplification, with a detection limit of 10 4 CFU / mL.

[0147] Fig. 9 middle, Fig. 9 a is the color development result of the Zn-NPs colorimetric immunochromatographic test strip in Example 5 for detecting PMB, with a quantitative range of 100-500 ng / mL, Fig. 9 b is the color development result of PMB detected by immunochromatographic test strip after chemical coordination signal amplification, and the detection limit is 100-1000ng / mL.

[0148] Fig.10 In Fig.10 As shown in a, in the common horseradish peroxidase (HRP)-catalyzed 3,3',5,5'-tetramethylbenzidine (TMB) color development reaction, the colored product oxidized TMB is very easy to diffuse on the NC membrane due to its good water solubility; Fig.10 As shown in b, in the coordination process based on metal ions, the ionic color developer is coordinated with the metal-based nanomaterials and fixed, and the resulting colored product does not diffuse.

[0149] Fig.11 In Fig.11 As shown in a, in the common metal in situ growth signal enhancement, colloidal gold is used as a substrate for silver reduction, but the silver nanoparticles are often reduced before reaching the T line and non-specifically adsorbed on the NC membrane, causing serious background interference in the T line reading range; Fig.11 As shown in b, in the metal ion-based coordination process, the ionic color developer will only react in situ with the metal nanoparticles fixed on the T line, and no colored substances will be generated at other locations of the NC membrane to cause interference.

[0150] Example 6 Second Immunochromatographic Kit for Detecting the Marker Carcinoembryonic Antigen CEA

[0151] 6.1 The immunochromatographic test kit for detecting the disease marker carcinoembryonic antigen of this embodiment includes independently set immunochromatographic test strips and a color developer;

[0152] like Fig.12As shown, the LFIA test strip includes a sample pad 1, a binding pad 2 embedded with a detection probe, a nitrocellulose membrane 5, an absorption pad 6 and a polyvinyl chloride backing substrate 7. The sample pad 1, the binding pad 2 embedded with a detection probe, the nitrocellulose membrane 5, and the absorption pad 6 are sequentially pasted on the polyvinyl chloride backing substrate 7, overlapping at adjacent locations, and the binding pad and the absorption pad overlap the nitrocellulose membrane by 3 mm respectively. The nitrocellulose membrane is provided with a detection line 3 and a quality control line 4, the detection line is sprayed with anti-carcinoembryonic antigen monoclonal antibody 1 (anti-CEA McAb1), and the quality control line is sprayed with a secondary antibody (sheep anti-mouse IgG).

[0153] The detection probe is Zn-NPs labeled with anti-carcinoembryonic antigen monoclonal antibody 2 (anti-CEA McAb2).

[0154] The color developing reagent is 200 μL of borate buffer with a pH of 8 to 9 containing 10 μg of zinc color developing reagent (20 μL is added dropwise when used).

[0155] The preparation method of the detection probe is the same as 3.3 in Example 3.

[0156] 6.2 Preparation of immunochromatographic test strips

[0157] It is basically the same as 1.2 in Example 1, except that the steps after step 3) are as follows:

[0158] 4) Preparation of a conjugate pad for embedding detection probes: first, soak a 16 mm wide polyester film in a conjugate pad treatment solution containing 5% sucrose, 5% BSA and 0.5% Tween-20 for 1-2 h, dry at 37° C., then spray the prepared detection probe on the conjugate pad at an amount of 5 μg / mm, and assemble it with the sample pad, nitrocellulose membrane and absorbent pad to prepare a test paper;

[0159] 5) Paste the nitrocellulose membrane onto a polyvinyl chloride backing substrate, and then paste the absorption pad, conjugate pad and sample pad in sequence from one side of the polyvinyl chloride backing substrate to the other side.

[0160] 6.3 Detection method:

[0161] The method for detecting carcinoembryonic antigen using the immunochromatographic kit of the present embodiment comprises the following steps: taking 60 μL of a CEA antigen sample to be tested diluted with a pH 7.4 phosphate buffer, dripping it onto the sample pad of the test strip, and letting it stand for 10 minutes. The colorimetric reading uses software to read the R value of the RGB of the T line of the test strip, and after the reading is completed, 20 μL of a signal enhancement solution containing a metal ion color developer is dripped onto the T line in situ, and the R value of the RGB of the T line of the test strip is read using software to achieve quantitative judgment of the CEA concentration in the sample to be tested.

[0162] like Fig.13As shown in FIG. 1 , the color development result of the Zn-NPs colorimetric immunochromatographic test strip with a conjugated pad in the detection kit of this embodiment is used to detect CEA. Compared with the test result without a conjugated pad, this method can also achieve the detection of 10 ng / mL CEA, as shown in FIG. Fig.13 b. After using chemical coordination signal amplification, the color of the test strip is significantly darker, proving that it also has a signal amplification effect. In addition, the signal products after signal amplification are concentrated on the T line without diffusion, and the NC membrane has no background interference, as shown in Fig.13 c, demonstrating the applicability of this signal amplification strategy.

[0163] The above are only embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A lateral flow immunochromatography kit based on metal ion chemical coordination signal amplification, characterized in that: The kit comprises independently arranged immunochromatographic test strips, detection probes and color developing agents; The immunochromatographic test strip comprises a sample pad, a nitrocellulose membrane, an absorption pad and a backing substrate, wherein the sample pad, the nitrocellulose membrane and the absorption pad are sequentially pasted on the backing substrate and overlapped at adjacent locations, a detection line and a quality control line are arranged on the nitrocellulose membrane, the detection line is coated with an anti-disease marker monoclonal antibody 1, an anti-pathogenic microorganism monoclonal antibody 1 or a small molecule to be tested, and a secondary antibody is sprayed on the quality control line; When the detection line is coated with anti-disease marker monoclonal antibody 1, the detection probe is a metal nanoparticle labeled with anti-disease marker monoclonal antibody 2; When the detection line is coated with anti-pathogenic microorganism monoclonal antibody 1, the detection probe is a metal nanoparticle labeled with anti-pathogenic microorganism monoclonal antibody 2; When the detection line is coated with a small molecule to be detected, the detection probe is a metal nanoparticle labeled with a monoclonal antibody against the small molecule to be detected.

2. A lateral flow immunochromatography kit based on metal ion chemical coordination signal amplification, characterized in that: The kit comprises an independently arranged immunochromatographic test strip and a color developer; The immunochromatographic test strip comprises a sample pad, a binding pad embedded with a detection probe, a nitrocellulose membrane, an absorption pad and a backing substrate. The sample pad, the binding pad embedded with the detection probe, the nitrocellulose membrane and the absorption pad are sequentially pasted on the backing substrate and overlapped at adjacent locations. A detection line and a quality control line are arranged on the nitrocellulose membrane. The detection line is coated with an anti-disease marker monoclonal antibody 1, an anti-pathogenic microorganism monoclonal antibody 1 or a small molecule to be detected, and a secondary antibody is sprayed on the quality control line. When the detection line is coated with anti-disease marker monoclonal antibody 1, the detection probe is a metal nanoparticle labeled with anti-disease marker monoclonal antibody 2; When the detection line is coated with anti-pathogenic microorganism monoclonal antibody 1, the detection probe is a metal nanoparticle labeled with anti-pathogenic microorganism monoclonal antibody 2; When the detection line is coated with a small molecule to be detected, the detection probe is a metal nanoparticle labeled with a monoclonal antibody against the small molecule to be detected.

3. The kit according to claim 1 or 2, characterized in that The disease marker is alpha-fetoprotein, the detection line is coated with an anti-alpha-fetoprotein monoclonal antibody 1, and the detection probe is Zr-NPs or Ca-NPs labeled with an anti-alpha-fetoprotein monoclonal antibody 2. The preparation method is as follows: a Zr-NPs or Ca-NPs solution with a concentration of 100 μg / mL is taken, and co-incubated with an anti-alpha-fetoprotein monoclonal antibody 2 with a concentration of 1 mg / mL, the mass ratio of the nanoparticles to the monoclonal antibody is 20:1, the incubation time is 1-2 hours, and then 1% bovine serum albumin is added to obtain labeled Zr-NPs or Ca-NPs and a detection probe with a concentration of 2 mg / mL.

4. The kit according to claim 1 or 2, characterized in that The disease marker is carcinoembryonic antigen; the detection line is coated with anti-carcinoembryonic antigen monoclonal antibody 1, and the detection probe is Zn-NPs labeled with anti-carcinoembryonic antigen monoclonal antibody 2. The preparation method is as follows: take a Zn-NPs solution with a concentration of 100 μg / mL, adjust the solution pH to 8-9, and co-incubate with anti-carcinoembryonic antigen monoclonal antibody 2 with a concentration of 1 mg / mL, the mass ratio of nanoparticles to monoclonal antibodies is 20:3, the incubation time is 4-6 hours, and then add 1% bovine serum albumin to obtain labeled Zn-NPs, and the concentration of the detection probe is 2 mg / mL.

5. The kit according to claim 1 or 2, characterized in that The pathogenic microorganism is Escherichia coli O157:H7; the detection line is coated with an anti-Escherichia coli O157:H7 monoclonal antibody 1, and the detection probe is Zn-NPs labeled with an anti-Escherichia coli O157:H7 monoclonal antibody 2. The preparation method is as follows: take a Zn-NPs solution with a concentration of 100 μg / mL, adjust the solution pH to 8-9, and co-incubate with an anti-Escherichia coli O157:H7 monoclonal antibody 2 with a concentration of 1 mg / mL, the mass ratio of nanoparticles to monoclonal antibodies is 20:3, the incubation time is 4-6 hours, and then add 1% bovine serum albumin to obtain labeled Zn-NPs, and the concentration of the detection probe is 2 mg / mL.

6. The kit according to claim 1 or 2, characterized in that The small molecule to be detected is polymyxin B, the detection line is coated with polymyxin B, and the detection probe is Zn-NPs labeled with anti-polymyxin B monoclonal antibody. The preparation method is as follows: take a Zn-NPs solution with a concentration of 100 μg / mL, adjust the solution pH to 8-9, and co-incubate with anti-polymyxin B monoclonal antibody with a concentration of 1 mg / mL, the mass ratio of nanoparticles to monoclonal antibodies is 20:3, the incubation time is 4-6 hours, and then add 1% bovine serum albumin to obtain labeled Zn-NPs, and the concentration of the detection probe is 2 mg / mL.

7. The kit according to claim 1 or 2, characterized in that The color developer is a color developer for Zr-NPs, a color developer for Ca-NPs or a color developer for Zn-NPs. The method for preparing the color developer for Zr-NPs is as follows: adding Zr ion color developer arsenazo into a 3M hydrochloric acid solution; the mass volume ratio of arsenazo to the hydrochloric acid solution is 10 μg:200 μL; The method for preparing the color developer for Ca-NPs is as follows: adding the Ca ion color developer arsenazo into a 0.01M phosphate buffer solution with a pH of 7.4; the mass volume ratio of arsenazo to the phosphate buffer solution is 10 μg:200 μL; The method for preparing the color developing agent for Zn-NPs is as follows: adding a zinc color developing agent into a borate buffer solution with a pH of 8 to 9; the mass volume ratio of the zinc color developing agent to the borate buffer solution is 10 μg:200 μL.

8. The kit according to claim 1, characterized in that The method for preparing the immunochromatographic test strip comprises the following steps: 1) Preparation and treatment of sample pad: cut the polyester fiber membrane into 20-25 mm width as the sample pad; soak it in sample pad treatment solution for 2-4 hours, and then bake it at 35-42°C for 1-3 hours; 2) Treatment of nitrocellulose membrane: first, prepare the anti-disease marker monoclonal antibody 1, the anti-pathogenic microorganism monoclonal antibody 1 or the small molecule to be tested into a 1 mg / mL spray solution, spray it on the nitrocellulose membrane to obtain the test line; then prepare the secondary antibody into a spray solution with a concentration of 1 mg / mL, spray it on the nitrocellulose membrane to obtain the quality control line; 3) Preparation of absorbent pad: Cut absorbent paper into 15-20 mm width as absorbent pad; 4) Paste the nitrocellulose membrane in step 2) onto a polyvinyl chloride backing substrate, and then paste the sample pad and the absorption pad in sequence from one side to the other side of the polyvinyl chloride backing substrate.

9. The kit according to claim 2, characterized in that The method for preparing the immunochromatographic test strip comprises the following steps: Steps 1)-3): the same as steps 1)-3) of claim 8; 4) Preparation of a conjugate pad for embedding detection probes: first, soak a 16 mm wide polyester film in a conjugate pad treatment solution for 1-2 h, dry it at 37° C., then spray the prepared detection probe on the conjugate pad at an amount of 5 μg / mm, and assemble it with a sample pad, a nitrocellulose membrane, and a water-absorbing pad to prepare a test paper; 5) Paste the nitrocellulose membrane onto a polyvinyl chloride backing substrate, and then paste the absorption pad, conjugate pad and sample pad in sequence from one side of the polyvinyl chloride backing substrate to the other side.

10. The kit according to claim 6 or 7, characterized in that The sample pad treatment solution contains 3-5wt% sucrose, 0.05-2wt% Triton-100, 0.05-0.1mol / L tris(hydroxymethyl)aminomethane hydrochloride, and 0.01M phosphate buffered saline solution; The conjugate pad treatment solution contains 5% sucrose, 5% BSA and 0.5% Tween-20.

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