Lateral flow chromatography test paper based on colorimetric signal amplification strategy for rapid deposition of enzyme-catalyzed polydopamine, preparation method and detection method thereof

Abstract

The invention discloses lateral flow chromatography test paper based on a colorimetric signal amplification strategy for rapid deposition of enzyme-catalyzed polydopamine, a preparation method and a detection method thereof, the lateral flow chromatography test paper forms an enzyme-labeled detection probe-target immune complex through an enzyme-labeled detection probe coated on a conjugate pad, and the enzyme-labeled detection probe-target immune complex is released from the conjugate pad; a sandwich immune sandwich compound is further formed on a detection line, a catalytic color developing solution is dropwise added to the detection line, a chromogenic substrate is green and efficient, the test paper is simple to assemble and convenient to use, qualitative and quantitative detection can be accurately completed, and high sensitivity and high specificity are achieved; joint detection of multiple target objects can be completed in a short time, the detection cost is greatly saved, the detection limit is reduced, and the method has general applicability in the field of target object detection.

Description

Technical Field

[0001] This invention relates to a side-flow chromatography test strip, and more particularly to a side-flow chromatography test strip based on a colorimetric signal amplification strategy of rapid polydopamine deposition catalyzed by enzymes. It also relates to a method for preparing the above-mentioned test strip and a detection method using the above-mentioned test strip. Background Technology

[0002] Point-of-care testing (POCT), with its advantages of speed, simplicity, and low overall cost, has been widely applied and demonstrated its clinical value in clinical settings, primary healthcare, public health control, and consumer self-testing. Lateral flow immunoassay (LFIA), a rapid on-site analytical method combining immunoassay and chromatographic techniques, has become a recognized mainstay of point-of-care diagnostic devices. Colloidal gold immunochromatography is the most common method, allowing for real-time colorimetric analysis with the naked eye, eliminating the need for a laboratory environment and professional operation, making it convenient to use. However, its relatively weak colorimetric signal intensity currently faces the challenge of insufficient detection sensitivity. Therefore, developing highly sensitive colorimetric signal amplification strategies for LFIA is of great practical significance. This not only ensures convenient and visualized signal readings but also provides a reliable basis for the analysis of trace targets using LFIA.

[0003] Enzyme-catalyzed signal generation and amplification technology has been widely used in LFIA (Low-Frequency Inhibitor-Amplifier). Currently, the common catalytic colorimetric signal amplification strategy in LFIA mainly relies on oxidoreductase-mediated catalytic chromogenic substrate reactions. Commonly used chromogenic substrates include 3-amino-N-ethylcarbazole (AEC), 4'-benzyltetramine (DAB), and 3,3',5,5'-tetramethylbenzidine (TMB). All of these require dissolution in organic solvents (such as acetone, dimethyl sulfoxide, etc.), must be prepared immediately, and require low-temperature storage. Leakage can easily contaminate the experimental environment. Furthermore, AEC and DAB have been proven to be carcinogenic and have potential mutagenic effects, posing a potential threat to the health of users, and have been gradually abandoned. TMB is currently the most widely used chromogenic substrate. However, because the oxidized TMB product generated by enzyme catalysis is a soluble molecule, it easily diffuses on the test paper, significantly reducing the accuracy and repeatability of signal reading. This greatly limits the development and practical application of enzyme-catalyzed colorimetric signal amplification technology. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a side-flow chromatography test strip that is green, efficient, highly sensitive, easy to operate, and low in cost, and can achieve the joint detection of multiple targets in a short time. This test strip is based on a colorimetric signal amplification strategy of rapid deposition of polydopamine catalyzed by enzymes. The invention also provides a method for preparing the test strip and a detection method using the test strip.

[0005] Technical Solution: The side-flow chromatography test paper of the present invention, based on a colorimetric signal amplification strategy of rapid polydopamine deposition catalyzed by enzymes, includes a polyvinyl chloride backing substrate and a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad sequentially pasted on one side of the polyvinyl chloride backing substrate. The sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad overlap at adjacent junctions. The conjugate pad is coated with an enzyme-labeled detection probe, and the nitrocellulose membrane is provided with a first detection line, a second detection line, and a control line. The enzyme-labeled detection probe includes natural horseradish peroxidase (HRP) or an immobilized enzyme.

[0006] The first and second detection lines are respectively coated with monoclonal capture antibodies corresponding to the two target analytes, and the control line is coated with secondary antibody. The first detection line, the second detection line, and the control line are perpendicular to the direction of liquid diffusion. The target substances include disease-related biomarkers, preferably alpha-fetoprotein and abnormal prothrombin.

[0007] The enzyme-labeled detection probe is a metal-organic framework immobilized horseradish peroxidase (HRP@MOF) labeled detection probe.

[0008] The method for preparing the above-mentioned side-flow chromatography test strip includes the following steps: A nitrocellulose membrane is adhered to a polyvinyl chloride (PVC) backing substrate. Then, from one side of the PVC backing substrate to the other side, a conjugate pad, a sample pad, and an absorbent pad are 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 strip is then dried at 35-42 ℃ for 1-3 hours.

[0009] The sample pads are pretreated as follows: the polyester film is cut into sample pads 20-30 mm wide, and 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 h, and then dried at 35-42 ℃ for 1-3 h.

[0010] The conjugate pad undergoes the following pretreatment: a polyester film is cut into conjugate pads 10-20 mm wide 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 then baked at 35-42°C for 1-3 h; the prepared enzyme-labeled detection probe 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 baked at 35-42°C for 1-3 h.

[0011] The nitrocellulose membrane undergoes the following pretreatment: a target monoclonal capture antibody spray solution with a concentration of 0.8~1.2 mg / mL is prepared and sprayed onto the nitrocellulose membrane in multiple intervals using a "scribing" method to obtain two detection lines 3~5 mm apart, with the first detection line and the second detection line sequentially from front to back, starting from the direction of the sample pad; a secondary antibody spray solution with a concentration of 0.8~1.2 mg / mL is prepared and sprayed onto the nitrocellulose membrane using a "scribing" method, controlling the distance from the second detection line to be 3~5 mm and the distance from the end of the nitrocellulose membrane to be 5~10 mm.

[0012] The preparation method of the enzyme-labeled detection probe for recognizing target 1 is as follows: take the enzyme solution, incubate it with the monoclonal antibody recognizing target 1 at room temperature, and then add bovine serum albumin to block it, thus obtaining the enzyme-labeled detection probe for recognizing target 1. The preparation method of the enzyme-labeled detection probe for recognizing target 2 is as follows: take the enzyme solution, incubate it with the monoclonal antibody recognizing target 2 at room temperature, and then add bovine serum albumin to block it, thus obtaining the enzyme-labeled detection probe for recognizing target 2. The concentration of the natural horseradish peroxidase (HRP) solution is 0.01~0.05 mg / mL, the concentration of the immobilized enzyme and nanozyme solution with peroxidase-like activity is 1~5 mg / mL, the concentration of the monoclonal capture antibody that recognizes target 1 is 1~3 mg / mL, and the incubation time is 4~6 h.

[0013] This invention also discloses a detection method using the above-mentioned side-flow chromatography test paper, comprising the following steps: (1) Prepare a catalytic colorimetric solution containing dopamine and hydrogen peroxide; (2) The sample to be tested is dropped onto the sample pad of the test strip. Under the action of capillary force, the sample solution migrates towards the nitrocellulose membrane. First, it recognizes and binds to the enzyme-labeled detection probe coated on the conjugate pad, forming an enzyme-labeled detection probe-target immune complex and is released from the conjugate pad. It further undergoes specific antigen-antibody recognition with the target monoclonal capture antibody on the first and second detection lines, thereby forming a sandwich immune sandwich complex on the detection lines. The enzyme-labeled detection probe is gathered on the first and second detection lines. After the control line develops color, the catalytic color development solution is dropped onto the detection line. (3) Qualitative analysis of the target substance is performed by visually observing the color depth of the detection line; or the RGB value of the detection line is analyzed using ColorPicker software, and the concentration of the target substance in the sample is quantified according to the standard curve.

[0014] Specifically, the catalytic colorimetric solution is prepared by thoroughly mixing 1-4 mL of an aqueous solution of dopamine hydrochloride with a concentration of 1-10 mg / mL with 0.5-2 mL of a hydrogen peroxide solution with a concentration of 0.1-1 M.

[0015] Invention Principle: This invention relates to a side-flow chromatography test strip based on a colorimetric signal amplification strategy for rapid polydopamine deposition catalyzed by enzymes. This strip can improve the detection sensitivity of LFIA. The enzyme-labeled probe used not only uses its own color to make the detection line appear colored, but also uses its enzyme activity to catalyze the rapid deposition of polydopamine, displaying the color of polydopamine with a strong colorimetric signal intensity. By observing the color intensity of the detection line with the naked eye, the concentration of the target substance can be qualitatively determined by colorimetry, greatly improving the overall portability, accuracy, and sensitivity of the detection.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The side-flow chromatographic test strip of the present invention based on the colorimetric signal amplification strategy of rapid deposition of polydopamine catalyzed by enzyme is green and efficient, the test strip is simple to assemble and easy to use, and can accurately complete qualitative and quantitative detection. It has high sensitivity and high specificity; it can complete the joint detection of multiple targets in a short time, which greatly saves detection costs and reduces the detection limit. It has universal applicability in the field of target detection; (2) The side-flow immunochromatographic test strip detection method based on the colorimetric signal amplification strategy of rapid deposition of polydopamine catalyzed by enzyme in the present invention, taking alpha-fetoprotein and abnormal prothrombin as examples, can detect target substances with a minimum concentration of 50 pg / mL and 100 pg / mL, respectively. All detections can be completed within 15 min. The test results can be observed with the naked eye. The present invention has strong specificity, high sensitivity, and is easy to use. The detection process is simple and fast, and the detection results are safe and reliable. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the side-flow chromatography test strip of the present invention, wherein 1 is the sample pad, 2 is the conjugation pad, 3 is the nitrocellulose membrane, 4 is the first detection line, 5 is the second detection line, 6 is the quality control line, 7 is the absorption pad, and 8 is the polyvinyl chloride plate. Figure 2 Hydration dynamic diameter (a) and surface potential diagram (b) of HRP@MOF-labeled detection probe; Figure 3 The UV-Vis absorption spectrum of the HRP@MOF catalytic chromogenic substrate; Figure 4 Images of polydopamine generated by HRP and HRP@MOF catalysis; Figure 5The colorimetric detection results of alpha-fetoprotein and abnormal prothrombin using HRP@MOF-based immunochromatographic test strips were visualized, with results of 5 ng / mL and 10 ng / mL, respectively. Figure 6 The standard curves for the detection results before signal amplification of rapid deposition of polydopamine are shown. In the figure, a is the standard curve for the detection results of alpha-fetoprotein and b is the standard curve for the detection results of abnormal prothrombin. Figure 7 The results of HRP@MOF-based immunochromatographic test strips for detecting alpha-fetoprotein and abnormal prothrombin after catalytic amplification were shown to be 50 pg / mL and 100 pg / mL, respectively. Figure 8 The standard curves for the detection results after signal amplification of rapid deposition of catalytic polydopamine are shown, where a is the standard curve for alpha-fetoprotein detection results and b is the standard curve for abnormal prothrombin detection results. Figure 9 The image shows the results of detecting the content of the target substance in serum. Detailed Implementation

[0018] The technical solution of the present invention will be further described below with reference to the embodiments. The test materials used in the embodiments can all be obtained through conventional means.

[0019] Example 1 The present invention relates to a side-flow chromatography test strip based on a colorimetric signal amplification strategy for rapid polydopamine deposition catalyzed by enzymes. The side-flow chromatography test strip includes a polyvinyl chloride backing substrate 8 and a sample pad 1, a conjugate pad 2, a nitrocellulose membrane 3, and an absorbent pad 7 sequentially pasted on one side plane of the polyvinyl chloride backing substrate 8. The sample pad 1, the conjugate pad 2, the nitrocellulose membrane 3, and the absorbent pad 7 overlap at adjacent junctions. The conjugate pad 2 is coated with an enzyme-labeled detection probe, and the nitrocellulose membrane is provided with a first detection line 4, a second detection line 5, and a control line 6.

[0020] The first detection line 4 and the second detection line 5 are respectively coated with monoclonal capture antibodies corresponding to the two targets, and the control line 6 is coated with secondary antibody. The first detection line 4, the second detection line 5 and the control line 6 are perpendicular to the direction of liquid diffusion. The targets include disease biomarkers, drugs or pathogenic microorganisms. The enzyme-labeled detection probes include natural horseradish peroxidase (HRP), immobilized enzymes or nanozymes with peroxidase-like activity.

[0021] Example 2 The preparation method of enzyme-labeled detection probes for recognizing target analytes is as follows: (1) Preparation of HRP@MOF: The MOF loaded with HRP used is zeolite imidazole ester framework material-8 (ZIF-8). First, 1~5 mmol of zinc nitrate and 0.01~0.05 mmol of 2-methylimidazolium are used as precursors, and 0.001~0.01 mmol of hexadecyltrimethylammonium bromide is used as surfactant. 50~80 mg of HRP is added to the system, and monodisperse ZIF-8 loaded with HRP is prepared by aqueous phase synthesis. After the reaction, it is HRP@MOF. (2) Preparation of HRP@MOF labeled detection probes: Take 1 mg / mL of HRP@MOF solution and incubate it with monoclonal antibodies that recognize target 1 and 2 at room temperature. Then add 10% bovine serum albumin to block it, and you will get HRP@MOF labeled detection probes that recognize target 1 and 2 respectively.

[0022] Example 3 This invention relates to a method for preparing lateral flow immunochromatography based on a colorimetric signal amplification strategy using enzyme-catalyzed rapid deposition of polydopamine, comprising the following steps: The LFIA test strip consists of four parts: a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad. The polyester membrane is cut into 20 mm wide sample pads and soaked in a solution of 3% sucrose, 0.05% Triton-100, 0.05 mol / L tris(hydroxymethyl)aminomethane hydrochloride (pH 8.0), and phosphate buffer for 2 h, then dried at 37 ℃ for 1 h. The polyester membrane is cut into 10 mm wide conjugate pads and treated with a phosphate buffer solution containing 3% sucrose, 5% bovine serum albumin, and 0.5% Tween-20 (pH 7.4), then dried at 37 ℃ for 1 h. The prepared HRP@MOF-labeled detection probes for identifying targets 1 and 2 are prepared as a 0.5 mg / mL spray solution and sprayed onto the treated conjugate pads using a "gold spray" method, then dried at 37 ℃ for 1 h. Absorbent paper is cut into 15 mm wide absorbent pads, which do not require further treatment. The sample pad, conjugation pad, nitrocellulose membrane, and absorbent pad are sequentially adhered to a polyvinyl chloride plate, overlapping each other by 2 mm, to assemble a side-flow chromatography test paper. Figure 1 As shown. The surface of the nitrocellulose membrane is provided with two detection lines and one control line. Monoclonal capture antibodies for the targets 1 and 2 to be detected are sprayed onto the nitrocellulose membrane at a rate of 1 μL / cm to form the first detection line and the second detection line, respectively. The secondary antibody is sprayed onto the nitrocellulose membrane at a rate of 1 μL / cm to form the control line.

[0023] Example 4 This invention relates to a detection method for lateral flow immunochromatography based on a colorimetric signal amplification strategy using enzyme-catalyzed rapid deposition of polydopamine, comprising the following steps: (1) Preparation of catalytic colorimetric solution: Weigh 50 mg of dopamine hydrochloride and dissolve it in 5 mL of ultrapure water to prepare a 10 mg / mL dopamine hydrochloride solution. Take 100 μL of 30% hydrogen peroxide solution and dilute it to 10 mL with water to prepare a 0.1 M hydrogen peroxide solution, which should be prepared and used immediately. Mix 4 mL of the 10 mg / mL dopamine hydrochloride solution with 2 mL of the 0.1 M hydrogen peroxide solution thoroughly to obtain the catalytic colorimetric solution; (2) Take 50 μL of the sample to be tested and drop it onto the sample pad of the test strip. The sample solution migrates to the nitrocellulose membrane under the action of capillary force. After the control line develops color, drop the catalytic color development solution prepared in step (4) onto the detection line. (3) After a period of time, the color depth of the detection line is observed by the naked eye to perform qualitative analysis of the target substance; or the RGB value of the detection line is measured by using ColorPicker software, and the concentration of the target substance in the sample is quantitatively analyzed according to the standard curve.

[0024] like Figure 2 The diagram shows the hydration kinetic diameter and surface potential of the HRP@MOF-labeled detection probe (HRP@MOF-Ab) of the present invention. As HRP is loaded into the MOF cavity to obtain HRP@MOF, and HRP@MOF surface is modified with detection antibody to obtain HRP@MOF-Ab, the hydration kinetic diameter gradually increases, while the surface potential gradually decreases, changing from positive to negative.

[0025] like Figure 3 The image shows the UV-Vis absorption spectrum of the HRP@MOF-catalyzed chromogenic substrate. In the 3,3',5,5'-tetramethylbenzidine (TMB)-hydrogen peroxide system, ultrapure water, natural enzyme HRP, immobilized enzyme HRP@MOF, and HRP@MOF-Ab were added. All three enzymes, including natural enzyme HRP, immobilized enzyme HRP@MOF, and HRP@MOF-Ab, catalyzed the generation of oxidized TMB, producing a characteristic absorption peak at 652 nm, demonstrating their good peroxidase activity.

[0026] like Figure 4 The image shown is a picture of the production of polydopamine catalyzed by HRP and HRP@MOF. In the dopamine (DA)-hydrogen peroxide system, ultrapure water, natural enzyme HRP, and HRP@MOF-Ab were added. Both natural enzyme HRP and HRP@MOF-Ab can catalyze the rapid production of brown-black polydopamine.

[0027] like Figure 5The image shows the colorimetric detection results of alpha-fetoprotein (AFP) and abnormal prothrombin using an HRP@MOF-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 <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 <10 ng / mL, the test strip does not show a color line.

[0028] like Figure 6 The figure shows the standard curves for detecting alpha-fetoprotein (AFP) and abnormal prothrombin before catalytic amplification using this immunochromatographic test strip platform. This indicates that the platform exhibits good linearity in detecting AFP and abnormal prothrombin, with a linear range of 5–500 ng / mL for quantitative detection of AFP. R0 2 =0.9924, the linear range for quantitative detection of abnormal prothrombin is 10~500 ng / mL, R 2 =0.9921.

[0029] like Figure 7 The results shown are the detection results of alpha-fetoprotein (AFP) and abnormal prothrombin after catalytic amplification using this immunochromatographic test strip platform. For AFP samples with a concentration of ≥50 pg / mL, the test strip shows a clear color line after detection, with a detection limit of 50 pg / mL; for AFP samples with a concentration less than 50 pg / mL, the test strip shows no color line. Compared to before catalytic amplification, the sensitivity for AFP detection has increased by 100 times. For abnormal prothrombin samples with a concentration of ≥100 pg / mL, the test strip shows a clear color line after detection, with a detection limit of 100 pg / mL; for abnormal prothrombin samples with a concentration less than 100 pg / mL, the test strip shows no color line. Compared to before catalytic amplification, the sensitivity for abnormal prothrombin detection has increased by 100 times.

[0030] like Figure 8 The figure shows the standard curves after amplification of the catalytic signals for detecting alpha-fetoprotein (AFP) and abnormal prothrombin using this immunochromatographic test strip platform. The linear range for quantitative detection of AFP is 0.05–10 ng / mL. 2 =0.9936, the linear range for quantitative detection of abnormal prothrombin is 0.1~10 ng / mL, R 2 =0.9895.

[0031] like Figure 9The results shown are those of the immunochromatographic test strip platform for detecting alpha-fetoprotein (AFP) and abnormal prothrombin in serum. Before signal amplification, the recovery rate of AFP was 93.63%–114.86% with a relative standard deviation (RSD) of 3.36%–8.38%, and the recovery rate of abnormal prothrombin was 93.60%–111.33% with an RSD of 5.90%–9.45%. After signal amplification, the recovery rate of AFP was 90.53%–110.31% with an RSD of 3.44%–9.30%, and the recovery rate of abnormal prothrombin was 94.16%–118.96% with an RSD of 3.60%–8.98%. This demonstrates the accuracy of this method for detecting AFP and abnormal prothrombin in blood samples.

Claims

1. A lateral flow chromatographic test strip based on colorimetric signal amplification strategy of rapid deposition of enzyme-catalyzed polydopamine, characterized in that, The lateral flow chromatographic test paper comprises a polyvinyl chloride backing substrate (8) and a sample pad (1), a conjugate pad (2), a nitrocellulose membrane (3) and an absorbent pad (7) sequentially pasted on one side of the polyvinyl chloride backing substrate (8), and the sample pad (1), the conjugate pad (2), the nitrocellulose membrane (3) and the absorbent pad (7) overlap at adjacent connecting portions; the conjugate pad (2) is coated with an enzyme-labeled detection probe, the nitrocellulose membrane is provided with a first detection line (4), a second detection line (5) and a quality control line (6), and the enzyme-labeled detection probe comprises natural horseradish peroxidase (HRP) or immobilized enzyme.

2. The lateral flow chromatographic test strip of claim 1, wherein, The first detection line (4) and the second detection line (5) are respectively sprayed with monoclonal capture antibodies corresponding to two target objects, and the quality control line (6) is sprayed with a secondary antibody; the first detection line (4), the second detection line (5) and the quality control line (6) are perpendicular to the liquid diffusion direction.

3. The lateral flow chromatographic test strip of claim 1, wherein, The enzyme-labeled detection probe is a metal-organic framework immobilized horseradish peroxidase (HRP)@MOF labeled detection probe.

4. The lateral flow chromatographic test strip of claim 2, wherein, The target objects comprise disease-related biomarkers.

5. A method of making the lateral flow chromatographic test strip of claim 1, wherein, The method comprises the following steps: pasting the nitrocellulose membrane on the polyvinyl chloride backing substrate, and then sequentially pasting the conjugate pad, the sample pad and the absorbent pad from one side to the other side of the polyvinyl chloride backing substrate, wherein the sample pad and the conjugate pad overlap by 2-4 mm, the conjugate pad and the absorbent pad overlap by 2-4 mm, and the nitrocellulose membrane overlaps by 2-4 mm, and the assembled product is placed in a drying oven at 35-42 ℃ for 1-3 h to obtain the lateral flow chromatographic test paper.

6. The method of claim 5, wherein, The sample pad is pretreated by the following method: cutting a polyester film into a sample pad with a width of 20-30 mm, and soaking the sample pad in a phosphate buffer solution containing 3-5% sucrose, 0.05-2% triton X-100 and 0.05-0.1 mol / L tris-hydroxymethyl aminomethane hydrochloride for 2-4 h, and then baking the sample pad at 35-42 ℃ for 1-3 h.

7. The manufacturing method according to claim 5, characterized in that, The conjugate pad is pretreated by the following method: cutting a polyester film into a conjugate pad with a width of 10-20 mm, and treating the conjugate pad with a phosphate buffer solution containing 3-5% sucrose, 5-20% bovine serum albumin and 0.5-5% tween-20, and then baking the conjugate pad at 35-42 ℃ for 1-3 h; preparing an enzyme-labeled detection probe into a spraying solution with a concentration of 0.1-1 mg / mL, and spraying the spraying solution on the treated conjugate pad by using a "gold spraying" mode, and then baking the conjugate pad at 35-42 ℃ for 1-3 h.

8. The manufacturing method according to claim 5, characterized in that, The nitrocellulose membrane is pretreated by the following method: preparing a monoclonal capture antibody spraying solution for target objects with a concentration of 0.8-1.2 mg / mL, and spraying the spraying solution on the nitrocellulose membrane by using a "membrane drawing" mode, so as to obtain two detection lines with a distance of 3-5 mm, and the first detection line and the second detection line are arranged in the order of the first detection line, the second detection line and the quality control line from the sample pad to the rear end of the nitrocellulose membrane; preparing a secondary antibody spraying solution with a concentration of 0.8-1.2 mg / mL, and spraying the spraying solution on the nitrocellulose membrane by using a "membrane drawing" mode, so as to control the distance between the secondary antibody and the second detection line to be 3-5 mm, and the distance between the secondary antibody and the end of the nitrocellulose membrane to be 5-10 mm.

9. A detection method using the lateral flow chromatographic test strip according to claim 1, characterized in that, The method comprises the following steps: (1) preparing a catalytic color developing solution containing dopamine and hydrogen peroxide; (2) The sample to be tested is added to the sample pad of the test strip, and the sample liquid migrates to the nitrocellulose membrane under the action of capillary force, first recognizes and combines with the enzyme-labeled detection probe coated on the binding pad to form an immune complex of the enzyme-labeled detection probe-target and is released from the binding pad, and then specifically recognizes the antigen-antibody on the target monoclonal capture antibody on the first detection line and the second detection line, thereby forming a sandwich immune complex on the detection line, and the enzyme-labeled detection probe is aggregated on the first detection line and the second detection line, and after the quality control line is colored, the catalytic color developing solution is added at the detection line; (3) The color depth on the detection line is observed by the naked eye to qualitatively analyze the target; or the RGB value of the detection line is analyzed by using ColorPicker software, and the concentration of the target in the sample to be tested is quantitatively analyzed according to a standard curve.

10. The detection method according to claim 9, characterized in that, The catalytic color developing solution is specifically prepared by fully mixing 1-4 mL of a dopamine hydrochloride aqueous solution with a concentration of 1-10 mg / mL and 0.5-2 mL of a hydrogen peroxide solution with a concentration of 0.1-1 M.

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