Preparation method of GDF-15 detection card, kit and use method thereof

Abstract

This invention discloses a method for preparing a GDF-15 detection card, its kit, and its usage. It uses a gold octahedral probe as a marker, the component of which is AuNO@4-MBA, where AuNO is a gold octahedron and @ indicates that the Raman signal molecule 4-mercaptobenzoic acid is modified on the AuNO surface. This invention uses a 4-MBA-labeled gold octahedral probe-labeled anti-GDF-15 monoclonal antibody. This immune complex is captured by another anti-GDF-15 monoclonal antibody immobilized on a nitrocellulose membrane. The intensity of the characteristic peak of the detection line is read using Raman spectroscopy or based on the colorimetric intensity. The sample detection has high sensitivity, and the positive or negative result can be directly determined based on the colorimetric intensity. It features a wide detection range, high sensitivity, high accuracy, and rapid and convenient detection, making it suitable for rapid detection.

Description

Technical Field

[0001] This invention relates to a method for preparing a GDF-15 detection card, belonging to the field of sample detection technology. Background Technology

[0002] Growth differentiation factor-15 (GDF-15) is an independent inflammatory biochemical marker reflecting cardiovascular function and disease. It is closely related to the occurrence and development of acute coronary syndrome (ACS) and has significant value in ACS risk stratification, providing decision support for long-term antithrombotic therapy in post-ACS patients. GDF-15 has high sensitivity and can be detected in the early stages of myocardial injury. Combined detection with traditional myocardial injury markers can improve the diagnostic ability for ACS. Currently, detection technologies for GDF-15 include enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence immunoassay (ECIA), fluorescence immunochromatography (FICI), and colloidal gold immunochromatography (FICI). ELISA and ECI require large, specialized instruments; colloidal gold immunochromatography is convenient and quick, allowing for self-testing, but its sensitivity is not high; fluorescence immunochromatography has high sensitivity and stability, but requires specialized instruments and cannot provide visualization. The patent application number 202111345701.5 addresses the issues of low detection range and insufficient sensitivity, but still requires the use of fluorescence equipment for processing, and a detection threshold remains. Therefore, it is of great significance to develop a chromatography kit and method with high sensitivity, rapid assay, high accuracy in detecting GDF-15 in serum, plasma and whole blood samples, and readability by the naked eye. Summary of the Invention

[0003] To solve the above technical problems, the present invention provides a method for preparing a GDF-15 detection card, comprising the following steps: S1: Preparation of binding pad: Add gold octahedral probe to GDF-15 monoclonal antibody 1, block with BSA, centrifuge at high speed, wash and resuspend with preservation solution, spray microspheres on the binding pad with GDF-15 monoclonal antibody 1 solution labeled with gold octahedral probe, and dry overnight. S2: Preparation of coating membrane: GDF-15 monoclonal antibody 2 and goat anti-mouse IgG antibody were used as the detection line and control line, respectively, and were drawn parallel to each other on the nitrocellulose membrane for coating. The membrane was then dried overnight. S3: Sample pad preparation: Soak the sample pad in sample pad treatment solution and dry it overnight; S4: On the substrate, the sample pad, bonding pad, coating film and absorbent paper are sequentially pasted together to obtain the test paper, which is then cut to obtain the GDF-15 test card.

[0004] Furthermore, the composition of the gold octahedral probe in step S1 is AuNO@4-MBA, where AuNO is a gold octahedron and @ indicates that the Raman signal molecule 4-mercaptobenzoic acid is modified on the AuNO surface.

[0005] Furthermore, in step S1, the amount of GDF-15 monoclonal antibody 1 added is 5~20µg / mL, and the BSA concentration is 20%.

[0006] Furthermore, in step S3, the sample pad treatment solution consists of 0.5% NaCl, 0.5% S17, 1% BSA, 20mM Tris-HCl with 1mg / ml anti-RBC antibody and pH 8.0.

[0007] Furthermore, in the detection line, the GDF-15 monoclonal antibody 2 coating concentration is 0.1~2 mg / ml and the volume is 0.5~1.5 µl of coating solution / cm membrane, and in the quality control line, the goat anti-mouse IgG antibody coating concentration is 0.5~2 mg / ml and the volume is 0.5~1.5 µl of coating solution / cm membrane.

[0008] Further, the gold octahedral probe was added to GDF-15 monoclonal antibody 1, blocked with BSA, centrifuged at high speed, washed and resuspended with preservation solution, and then diluted 4-10 times with microsphere dilution buffer to obtain a solution of gold octahedral probe-labeled GDF-15 monoclonal antibody 1. The microsphere dilution buffer consisted of 0.5% Tween-20, 2% BSA, 5% sucrose, and 20mM, pH 8.0 PBS.

[0009] Furthermore, the present invention also provides a method for preparing the gold octahedral probe, which includes the following steps: A1: Synthesis of gold nanorods: Chloroauric acid was added to hexadecyltrimethylammonium bromide (CTAB) under magnetic stirring, sodium borohydride solution was added to dissolve, and the mixture was stirred and left to stand for 30 min; A2: Preparation of growth medium: CTAB and sodium oleate were dissolved in ultrapure water, chloroauric acid solution was added, and silver nitrate solution was added after the solution became colorless. After stirring, hydrochloric acid and ascorbic acid were added. After the reaction, seed solution was added. The reaction mixture was allowed to stand for 12 hours, centrifuged and washed, and then dispersed in ultrapure water. A3: Synthesis of gold nano octahedrons: CTAB and chloroauric acid were mixed and heated in a 60°C water bath. 3-Butenoic acid was added. After the reaction became colorless, gold nanorods were added and the mixture was allowed to stand in a 60°C water bath for 12 hours to obtain a gold octahedron solution. A4: Modification of Raman signal molecules: Add 4-mercaptobenzoic acid (4-MBA) solution to the gold octahedral solution, let stand for 4 hours, centrifuge to remove excess 4-MBA, and resuspend in pure water.

[0010] The present invention also provides a GDF-15 detection kit, wherein the detection card is made using the above-described method for preparing the GDF-15 detection card.

[0011] The method of using the GDF-15 test kit is as follows: 80 μL of sample is applied to the sample pad. After 15 minutes, the sample is visually interpreted, and a portable Raman spectrometer with a 785 nm laser is used to acquire the Raman signal of the test strip's detection line at a laser power of 8.4 mW, an integration time of 5 s, and two cumulative counts. During the test, BWSpec4.0 software is used to baseline and smooth the raw data for noise reduction, and Origin software is used to perform curve fitting on the processed data.

[0012] By adopting the above technical solution, the present invention has the following beneficial effects: (1) Using a monoclonal antibody against GDF-15 labeled with a gold octahedral probe labeled with 4-MBA, the immune complex will be captured by another monoclonal antibody against GDF-15 immobilized on a nitrocellulose membrane. The characteristic peak intensity of the detection line can be read by Raman spectroscopy or by color intensity. The sample detection sensitivity is high, and the positive and negative results can be directly determined by color development. It has the characteristics of wide detection range, high sensitivity, high accuracy, fast and simple detection, and can be used for rapid detection.

[0013] (2) This invention uses gold nanorods, which are relatively easy to prepare, as the core to grow gold nano octahedrons. It can easily prepare a metal substrate with extremely strong Raman enhancement effect, and the manufacturing process is more stable and convenient, making the industrialization of highly sensitive Raman test strips possible.

[0014] (3) The nanoprobe used in this invention is a gold octahedral probe, which has surface-enhanced Raman scattering (SERS).

[0015] Compared with traditional bioanalytical methods, SERS has unique advantages in bioanalysis, including: high sensitivity; resistance to photobleaching and photodegradation compared to fluorescence, making it suitable for long-term monitoring; and narrow spectral bandwidth, with Raman characteristic peaks typically being very narrow, 10-100 times narrower than the fluorescence emission peaks of organic dyes or quantum dots. Attached Figure Description

[0016] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein... Figure 1 Transmission electron microscope image of a gold octahedral probe; Figure 2 This is a schematic diagram of the structure of the GDF-15 detection card of the present invention; Figure 3 The standard curve of the average value of the Raman characteristic peaks; Figure 4 Linear analysis of concentration detection results.

[0017] Figure 5 This is a photograph of the test strip as observed with the naked eye.

[0018] The attached figures are labeled as follows: substrate 1, coating membrane 2, detection line 21, quality control line 22, conjugate pad 3, sample pad 4, absorbent paper 5. Detailed Implementation

[0019] To better understand the above technical solutions, the following will provide a detailed explanation of the technical solutions in conjunction with the accompanying drawings and specific implementation methods.

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0021] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0022] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0023] In the description of the embodiments of the present invention, it should be understood that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship commonly used when the product of the invention is in use, or the orientation or positional relationship commonly understood by those skilled in the art. They are only used to facilitate the description of the present invention and to simplify the description, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the present invention.

[0024] In the description of the embodiments of the present invention, it should also be noted that, unless otherwise explicitly specified and limited, the terms "set," "install," "connect," and "link" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal communication between two components. Those skilled in the art can understand the specific meaning of the above terms in the present invention based on the specific circumstances. The present invention will be further described below with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention and should not be used to limit the scope of protection of the present invention.

[0025] (Example 1) The preparation method of a GDF-15 detection card in this embodiment includes the following steps: S1: Preparation of binding pad 3: Add GDF-15 monoclonal antibody 1 to the gold octahedral probe at 20 µg / mL, mix well, let stand for 10 min, add 10 μl / mL of 20% BSA for blocking, mix well, let stand for 5 min, centrifuge at 10000 rpm for 10 min, wash with 20 mM, pH 8.0 PBS buffer containing 1% BSA and resuspend to 20% of the original volume, and spray a line evenly on the binding pad with GDF-15 monoclonal antibody 1 labeled with gold octahedral probe diluted 4 times with microsphere dilution buffer containing 0.5% Tween-20, 2% BSA, 5% sucrose, 20 mM, pH 8.0 PBS buffer, using 2 µl liquid volume / cm sample pad, place in an oven and dry at 37°C overnight; S2: Preparation of coating membrane 2: GDF-15 monoclonal antibody 2 and goat anti-mouse IgG antibody were used as detection lines and control lines, respectively, and were drawn parallel to each other on the nitrocellulose membrane for coating. The membrane was then dried. The concentrations of GDF-15 monoclonal antibody 2 and goat anti-mouse IgG antibody were adjusted to 0.5 mg / ml with 10 mM PB coating solution containing 2.5% trehalose. The amount of coating solution used was 0.5 µl / cm of membrane. The membrane was drawn parallel to each other as detection lines and control lines, respectively, with a 3 mm gap between the control lines and the detection lines. The membrane was then placed in an oven and dried at 45°C overnight. S3: Sample pad preparation 4: Spray two parallel lines evenly on one side of the conjugate pad with a sample pad treatment solution containing 0.5% NaCl, 0.5% S17, 1% BSA, 1 mg / ml anti-RBC antibody in 20mM Tris-HCl at pH 8.0. The amount of solution used is 2~4µl / cm. Dry at 37 degrees overnight. S4: On the base liner 1 (size 80*300mm), the sample pad 4 (size 23*300mm, made of glass fiber cotton), the bonding pad 3 (size 10*300mm, made of glass fiber cotton), the coating film 2 (size 25*300mm, made of nitrocellulose) and the absorbent paper 5 (size 28*300mm) are sequentially overlapped to obtain the test paper, and then cut it into 4mm wide GDF-15 test cards as required.

[0026] The amount of GDF-15 monoclonal antibody 1 labeled with gold octahedral probe sprayed on the conjugate pad 2 is 5~20µg antibody / mL gold octahedral probe.

[0027] The test line was coated with GDF-15 monoclonal antibody 2 (1 mg / ml), and the control line was coated with goat anti-mouse IgG antibody at a concentration of 1 mg / ml (the GDF-15 monoclonal antibody was purchased from Nanjing Baikang Biotechnology Co., Ltd., and the goat anti-mouse IgG antibody was from Changsha Boyou Biotechnology Co., Ltd.). The volume of coating solution used for the microsphere line was 4 µl / cm sample pad, and the volume of coating solution used for the test line and control line was 1 µl / cm membrane.

[0028] The method for preparing the gold octahedral probe includes: A1: Synthesis of gold nanorods: In a container, 250 µL of 10 mM chloroauric acid was added to 9.75 mL of 0.1 M hexadecyltrimethylammonium bromide (CTAB) under magnetic stirring, followed by the addition of 0.6 mL of 10 mM sodium borohydride solution. After stirring for 2 min, the mixture was placed at 28 °C for 30 min. A2: Growth solution: 7.2 g CTAB and 0.987 g sodium oleate were dissolved in 400 mL of ultrapure water. Then, 20 mL of 10 mM chloroauric acid was injected into the solution. When the solution became colorless, 8 mL of 10 mM silver nitrate solution was added. After stirring for 5 min, 1.2 mL of hydrochloric acid and 600 µL of 0.1 M ascorbic acid were added. After reacting for about 30 s, 1 mL of seed solution was added. The reaction mixture was allowed to stand at 30 °C for 12 hours to complete the synthesis of gold nanorods. After synthesis, the nanorods were centrifuged at 9000 rpm for 15 min, washed twice, and dispersed in 20 mL of ultrapure water. A3: Synthesis of gold nano octahedrons: 50 mL of 10 mM CTAB and 500 µL of 0.05 M chloroauric acid were mixed and heated in a 60 °C water bath for 2 min. Then, 220 µL of 3-butenoic acid was added to the mixture. When the solution became colorless, 300 µL of gold nanorods were added and the mixture was allowed to stand in a 60 °C water bath for 12 hours. A4: Modification of Raman signal molecules: Take 5 mL of gold octahedral solution and add 250 μL of 10 -4The 4-mercaptobenzoic acid (4-MBA) solution of M was allowed to stand for 4 hours, then centrifuged to remove excess 4-MBA, and then resuspended in pure water. This completed the modification of the Raman signal molecule, resulting in AuNO@4-MBA.

[0029] A GDF-15 detection kit, wherein the detection card is prepared using the above-mentioned method for preparing GDF-15 detection cards.

[0030] The method of using the above-mentioned GDF-15 test kit is as follows: 80 μL of sample is applied to the sample pad. After 15 minutes, the sample is visually interpreted, and a portable Raman spectrometer with a 785 nm laser is used to acquire the Raman signal of the test strip's detection line at a laser power of 8.4 mW, an integration time of 5 seconds, and two cumulative summations. During the test, BWSpec4.0 software is used to baseline and smooth the raw data for noise reduction, and Origin software is used to perform curve fitting on the processed data. (Example 2) The preparation method of a GDF-15 detection card in this embodiment includes the following steps: S1: Preparation of binding pad 3: Add GDF-15 monoclonal antibody 1 to the gold octahedral probe at 20 µg / mL, mix well, let stand for 10 min, add 10 μl / mL of 20% BSA for blocking, mix well, let stand for 5 min, centrifuge at 10000 rpm for 10 min, wash with 20 mM PBS buffer containing 1% BSA, pH 8.0 and resuspend to 20% of the original volume, spray a line evenly on the binding pad with GDF-15 monoclonal antibody 1 labeled with gold octahedral probe diluted 10 times with 20 mM PBS buffer containing 0.5% Tween-20, 2% BSA, and 5% sucrose, at a volume of 4 µl / cm sample pad, place in an oven and dry at 37°C overnight; S2: Preparation of coating membrane 2: GDF-15 monoclonal antibody 2 and goat anti-mouse IgG antibody were used as detection lines and control lines, respectively, and were streaked parallel to each other on the nitrocellulose membrane for coating. The membrane was then dried. The concentrations of GDF-15 monoclonal antibody 2 and goat anti-mouse IgG antibody were adjusted to 2 mg / ml with 10 mM PB coating solution containing 2.5% trehalose. The amount of coating solution used was 1.5 µl / cm of membrane. The membrane was streaked parallel to each other on the nitrocellulose membrane as detection lines and control lines, respectively, with a 7 mm gap between the control lines and the detection lines. The membrane was then placed in an oven and dried at 45°C overnight. S3: Sample pad preparation 4: Spray two parallel lines evenly on one side of the conjugate pad with a sample pad treatment solution containing 0.5% NaCl, 0.5% S17, 1% BSA, 1 mg / ml anti-RBC antibody in 20mM Tris-HCl at pH 8.0. The amount of solution used is 2~4µl / cm. Dry at 37 degrees overnight. S4: On the base liner 1 (size 80*300mm), the sample pad 4 (size 23*300mm, made of glass fiber cotton), the bonding pad 3 (size 10*300mm, made of glass fiber cotton), the coating film 2 (size 25*300mm, made of nitrocellulose) and the absorbent paper 5 (size 28*300mm) are sequentially overlapped to obtain the test paper, and then cut it into 4mm wide GDF-15 test cards as required.

[0031] The amount of GDF-15 monoclonal antibody 1 labeled with gold octahedral probe sprayed on the conjugate pad 2 is 20 µg antibody / mL gold octahedral probe.

[0032] The detection line was coated with GDF-15 monoclonal antibody 2 (2 mg / ml), and the control line was coated with goat anti-mouse IgG antibody at a concentration of 2 mg / ml (the GDF-15 monoclonal antibody was purchased from Nanjing Baikang Biotechnology Co., Ltd., and the goat anti-mouse IgG antibody was from Changsha Boyou Biotechnology Co., Ltd.). The volume of coating solution used for the microsphere line was 4 µl / cm sample pad, and the volume of coating solution used for the detection line and control line was 1.5 µl / cm membrane.

[0033] The method for preparing the gold octahedral probe includes: A1: Synthesis of gold nanorods: In a container, 250 µL of 10 mM chloroauric acid was added to 9.75 mL of 0.1 M hexadecyltrimethylammonium bromide (CTAB) under magnetic stirring, followed by the addition of 0.6 mL of 10 mM sodium borohydride solution. After stirring for 2 min, the mixture was placed at 28 °C for 30 min. A2: Growth solution: 7.2 g CTAB and 0.987 g sodium oleate were dissolved in 400 mL of ultrapure water. Then, 20 mL of 10 mM chloroauric acid was injected into the solution. When the solution became colorless, 8 mL of 10 mM silver nitrate solution was added. After stirring for 5 min, 1.2 mL of hydrochloric acid and 600 µL of 0.1 M ascorbic acid were added. After reacting for about 30 s, 1 mL of seed solution was added. The reaction mixture was allowed to stand at 30 °C for 12 hours to complete the synthesis of gold nanorods. After synthesis, the nanorods were centrifuged at 9000 rpm for 15 min, washed twice, and dispersed in 20 mL of ultrapure water. A3: Synthesis of gold nano octahedrons: 50 mL of 10 mM CTAB and 500 µL of 0.05 M chloroauric acid were mixed and heated in a 60 °C water bath for 2 min. Then, 220 µL of 3-butenoic acid was added to the mixture. When the solution became colorless, 300 µL of gold nanorods were added and the mixture was allowed to stand in a 60 °C water bath for 12 hours. A4: Modification of Raman signal molecules: Take 5 mL of gold octahedral solution and add 250 μL of 10 -4 The 4-mercaptobenzoic acid (4-MBA) solution of M was allowed to stand for 4 hours, then centrifuged to remove excess 4-MBA, and then resuspended in pure water. This completed the modification of the Raman signal molecule, resulting in AuNO@4-MBA.

[0034] A GDF-15 detection kit, wherein the detection card is prepared using the above-mentioned method for preparing GDF-15 detection cards.

[0035] The method of using the GDF-15 test kit is as follows: 80 μL of sample is applied to the sample pad. After 15 minutes, the sample is visually interpreted, and a portable Raman spectrometer with a 785 nm laser is used to acquire the Raman signal of the test strip's detection line with a laser power of 8.4 mW, an integration time of 5 s, and two cumulative counts. During the test, BWSpec4.0 software is used to baseline and smooth the raw data for noise reduction, and Origin software is used to perform curve fitting on the processed data.

[0036] (Example 3) The preparation method of a GDF-15 detection card in this embodiment includes the following steps: S1: Preparation of binding pad 3: Add GDF-15 monoclonal antibody 1 to the gold octahedral probe at 20 µg / mL, mix well, let stand for 10 min, add 10 μl / mL of 20% BSA for blocking, mix well, let stand for 5 min, centrifuge at 10000 rpm for 10 min, wash with 20 mM, pH 8.0 PBS buffer containing 1% BSA and resuspend to 20% of the original volume, and spray a line evenly on the binding pad with GDF-15 monoclonal antibody 1 labeled with gold octahedral probe diluted 4 times with microsphere dilution buffer containing 0.5% Tween-20, 2% BSA, 5% sucrose, 20 mM, pH 8.0 PBS buffer, using 4 µl liquid volume / cm sample pad, place in an oven and dry at 37°C overnight; S2: Preparation of coating membrane 2: GDF-15 monoclonal antibody 2 and goat anti-mouse IgG antibody were used as detection lines and control lines, respectively, and were streaked parallel to each other on the nitrocellulose membrane for coating. The membrane was then dried. The concentrations of GDF-15 monoclonal antibody 2 and goat anti-mouse IgG antibody were adjusted to 0.5 mg / ml with 10 mM PB coating solution containing 2.5% trehalose. The amount of coating solution used was 1.5 µl / cm of membrane. The membrane was streaked parallel to each other on the nitrocellulose membrane as detection lines and control lines, respectively, with a 3 mm gap between the control lines and the detection lines. The membrane was then placed in an oven and dried at 45°C overnight. S3: Sample pad preparation 4: Spray two parallel lines evenly on one side of the conjugate pad with a sample pad treatment solution containing 0.5% NaCl, 0.5% S17, 1% BSA, 1 mg / ml anti-RBC antibody in 20mM Tris-HCl at pH 8.0. The amount of solution used is 2~4µl / cm. Dry at 37 degrees overnight. S4: On the base liner 1 (size 80*300mm), the sample pad 4 (size 23*300mm, made of glass fiber cotton), the bonding pad 3 (size 10*300mm, made of glass fiber cotton), the coating film 2 (size 25*300mm, made of nitrocellulose) and the absorbent paper 5 (size 28*300mm) are sequentially overlapped to obtain the test paper, and then cut it into 4mm wide GDF-15 test cards as required.

[0037] The sample pad 4 is coated with microspheres and the gold octahedral probe is labeled with GDF-15 monoclonal antibody 1. The coating membrane 2 is provided with a detection line 21 and a control line 22. The detection line 21 is close to the binding pad 3, and the detection line 21 and the control line 22 are parallel to each other and spaced 5 mm apart. The detection line 21 is coated with GDF-15 monoclonal antibody 2, and the control line 22 is coated with goat anti-mouse IgG antibody.

[0038] The amount of GDF-15 monoclonal antibody 1 labeled with gold octahedral probe sprayed on the conjugate pad 2 is 5 µg antibody / mL gold octahedral probe.

[0039] The detection line was coated with GDF-15 monoclonal antibody 2 (0.5 mg / ml), and the control line was coated with goat anti-mouse IgG antibody at a concentration of 0.5 mg / ml (the GDF-15 monoclonal antibody was purchased from Nanjing Baikang Biotechnology Co., Ltd., and the goat anti-mouse IgG antibody was from Changsha Boyou Biotechnology Co., Ltd.). The volume of coating solution used for the microsphere line was 4 µl / cm sample pad, and the volume of coating solution used for the detection line and control line was 1.5 µl / cm membrane.

[0040] The method for preparing the gold octahedral probe includes: A1: Synthesis of gold nanorods: In a container, 250 µL of 10 mM chloroauric acid was added to 9.75 mL of 0.1 M hexadecyltrimethylammonium bromide (CTAB) under magnetic stirring, followed by the addition of 0.6 mL of 10 mM sodium borohydride solution. After stirring for 2 min, the mixture was placed at 28 °C for 30 min. A2: Growth solution: 7.2 g CTAB and 0.987 g sodium oleate were dissolved in 400 mL of ultrapure water. Then, 20 mL of 10 mM chloroauric acid was injected into the solution. When the solution became colorless, 8 mL of 10 mM silver nitrate solution was added. After stirring for 5 min, 1.2 mL of hydrochloric acid and 600 µL of 0.1 M ascorbic acid were added. After reacting for about 30 s, 1 mL of seed solution was added. The reaction mixture was allowed to stand at 30 °C for 12 hours to complete the synthesis of gold nanorods. After synthesis, the nanorods were centrifuged at 9000 rpm for 15 min, washed twice, and dispersed in 20 mL of ultrapure water. A3: Synthesis of gold nano octahedrons: 50 mL of 10 mM CTAB and 500 µL of 0.05 M chloroauric acid were mixed and heated in a 60 °C water bath for 2 min. Then, 220 µL of 3-butenoic acid was added to the mixture. When the solution became colorless, 300 µL of gold nanorods were added and the mixture was allowed to stand in a 60 °C water bath for 12 hours. A4: Modification of Raman signal molecules: Take 5 mL of gold octahedral solution and add 250 μL of 10 -4 The 4-mercaptobenzoic acid (4-MBA) solution of M was allowed to stand for 4 hours, then centrifuged to remove excess 4-MBA, and then resuspended in pure water. This completed the modification of the Raman signal molecule, resulting in AuNO@4-MBA.

[0041] A GDF-15 detection kit, wherein the detection card is prepared using the above-mentioned method for preparing GDF-15 detection cards.

[0042] The method of using the GDF-15 test kit is as follows: 80 μL of sample is applied to the sample pad. After 15 minutes, the sample is visually interpreted, and a portable Raman spectrometer with a 785 nm laser is used to acquire the Raman signal of the test strip's detection line with a laser power of 8.4 mW, an integration time of 5 s, and two cumulative counts. During the test, BWSpec4.0 software is used to baseline and smooth the raw data for noise reduction, and Origin software is used to perform curve fitting on the processed data.

[0043] (Example 4) The preparation method of a GDF-15 detection card in this embodiment includes the following steps: S1: Preparation of binding pad 3: Add GDF-15 monoclonal antibody 1 to the gold octahedral probe at 20 µg / mL, mix well, let stand for 10 min, add 10 μl / mL of 20% BSA for blocking, mix well, let stand for 5 min, centrifuge at 10000 rpm for 10 min, wash with 20 mM PBS buffer containing 1% BSA, pH 8.0 and resuspend to 20% of the original volume, spray a line evenly on the binding pad with GDF-15 monoclonal antibody 1 labeled with gold octahedral probe diluted 4 times with 20 mM PBS buffer containing 0.5% Tween-20, 2% BSA, and 5% sucrose, using a liquid volume of 4 µl / cm sample pad, place in an oven and dry at 37°C overnight; S2: Preparation of coating membrane 2: GDF-15 monoclonal antibody 2 and goat anti-mouse IgG antibody were used as detection lines and control lines, respectively, and were drawn parallel to each other on the nitrocellulose membrane for coating. The membrane was then dried. The concentrations of GDF-15 monoclonal antibody 2 and goat anti-mouse IgG antibody were adjusted to 0.5 mg / ml with 10 mM PB coating solution containing 2.5% trehalose. The amount of coating solution used was 0.5 µl / cm of membrane. The membrane was drawn parallel to each other as detection lines and control lines, respectively, with a 7 mm gap between the control lines and the detection lines. The membrane was then placed in an oven and dried at 45°C overnight. S3: Sample pad preparation 4: Spray two parallel lines evenly on one side of the conjugate pad with a sample pad treatment solution containing 0.5% NaCl, 0.5% S17, 1% BSA, 1 mg / ml anti-RBC antibody in 20mM Tris-HCl at pH 8.0. The amount of solution used is 2~4µl / cm. Dry at 37 degrees overnight. S4: On the base liner 1 (size 80*300mm), the sample pad 4 (size 23*300mm, made of glass fiber cotton), the bonding pad 3 (size 10*300mm, made of glass fiber cotton), the coating film 2 (size 25*300mm, made of nitrocellulose) and the absorbent paper 5 (size 28*300mm) are sequentially overlapped to obtain the test paper, and then cut it into 4mm wide GDF-15 test cards as required.

[0044] The sample pad 4 is coated with microspheres and the gold octahedral probe is labeled with GDF-15 monoclonal antibody 1. The coating membrane 2 is provided with a detection line 21 and a control line 22. The detection line 21 is close to the binding pad 3, and the detection line 21 and the control line 22 are parallel to each other and spaced 7 mm apart. The detection line 21 is coated with GDF-15 monoclonal antibody 2, and the control line 22 is coated with goat anti-mouse IgG antibody.

[0045] The amount of GDF-15 monoclonal antibody 1 labeled with gold octahedral probe sprayed on the conjugate pad 2 is 20 µg antibody / mL gold octahedral probe.

[0046] The detection line was coated with GDF-15 monoclonal antibody 2 (0.5 mg / ml), and the control line was coated with goat anti-mouse IgG antibody at a concentration of 0.5 mg / ml (the GDF-15 monoclonal antibody was purchased from Nanjing Baikang Biotechnology Co., Ltd., and the goat anti-mouse IgG antibody was from Changsha Boyou Biotechnology Co., Ltd.). The volume of coating solution used for the microsphere line was 4 µl / cm sample pad, and the volume of coating solution used for the detection line and control line was 0.5 µl / cm membrane.

[0047] The method for preparing the gold octahedral probe includes: A1: Synthesis of gold nanorods: In a container, 250 µL of 10 mM chloroauric acid was added to 9.75 mL of 0.1 M hexadecyltrimethylammonium bromide (CTAB) under magnetic stirring, followed by the addition of 0.6 mL of 10 mM sodium borohydride solution. After stirring for 2 min, the mixture was placed at 28 °C for 30 min. A2: Growth solution: 7.2 g CTAB and 0.987 g sodium oleate were dissolved in 400 mL of ultrapure water. Then, 20 mL of 10 mM chloroauric acid was injected into the solution. When the solution became colorless, 8 mL of 10 mM silver nitrate solution was added. After stirring for 5 min, 1.2 mL of hydrochloric acid and 600 µL of 0.1 M ascorbic acid were added. After reacting for about 30 s, 1 mL of seed solution was added. The reaction mixture was allowed to stand at 30 °C for 12 hours to complete the synthesis of gold nanorods. After synthesis, the nanorods were centrifuged at 9000 rpm for 15 min, washed twice, and dispersed in 20 mL of ultrapure water. A3: Synthesis of gold nano octahedrons: 50 mL of 10 mM CTAB and 500 µL of 0.05 M chloroauric acid were mixed and heated in a 60 °C water bath for 2 min. Then, 220 µL of 3-butenoic acid was added to the mixture. When the solution became colorless, 300 µL of gold nanorods were added and the mixture was allowed to stand in a 60 °C water bath for 12 hours. A4: Modification of Raman signal molecules: Take 5 mL of gold octahedral solution and add 250 μL of 10 -4 The 4-mercaptobenzoic acid (4-MBA) solution of M was allowed to stand for 4 hours, then centrifuged to remove excess 4-MBA, and then resuspended in pure water. This completed the modification of the Raman signal molecule, resulting in AuNO@4-MBA.

[0048] A GDF-15 detection kit, wherein the detection card is prepared using the above-mentioned method for preparing GDF-15 detection cards.

[0049] The method of using the GDF-15 test kit is as follows: 80 μL of sample is applied to the sample pad. After 15 minutes, the sample is visually interpreted, and a portable Raman spectrometer with a 785 nm laser is used to acquire the Raman signal of the test strip's detection line with a laser power of 8.4 mW, an integration time of 5 s, and two cumulative counts. During the test, BWSpec4.0 software is used to baseline and smooth the raw data for noise reduction, and Origin software is used to perform curve fitting on the processed data.

[0050] (Example 5) The preparation method of a GDF-15 detection card in this embodiment includes the following steps: S1: Preparation of binding pad 3: Add GDF-15 monoclonal antibody 1 to the gold octahedral probe at 20 µg / mL, mix well, let stand for 10 min, add 10 μl / mL of 20% BSA for blocking, mix well, let stand for 5 min, centrifuge at 10000 rpm for 10 min, wash with 20 mM PBS buffer containing 1% BSA, pH 8.0 and resuspend to 20% of the original volume, and spray a line evenly on the binding pad with GDF-15 monoclonal antibody 1 labeled with gold octahedral probe diluted 7 times with microsphere dilution buffer containing 0.5% Tween-20, 2% BSA, 5% sucrose, 20 mM, pH 8.0 PBS, using 2 µl liquid volume / cm sample pad, place in an oven and dry at 37°C overnight.

[0051] S2: Preparation of coating membrane 2: GDF-15 monoclonal antibody 2 and goat anti-mouse IgG antibody were used as detection lines and control lines, respectively, and were streaked parallel to each other on the nitrocellulose membrane for coating. The membrane was then dried. The concentrations of GDF-15 monoclonal antibody 2 and goat anti-mouse IgG antibody were adjusted to 2 mg / ml with 10 mM PB coating solution containing 2.5% trehalose. The amount of coating solution used was 0.5 µl / cm of membrane. The membrane was streaked parallel to each other on the nitrocellulose membrane as detection lines and control lines, respectively, with a 5 mm gap between the control lines and the detection lines. The membrane was then placed in an oven and dried at 45°C overnight. S3: Sample pad preparation 4: Spray two parallel lines evenly on one side of the conjugate pad with a sample pad treatment solution containing 0.5% NaCl, 0.5% S17, 1% BSA, 1 mg / ml anti-RBC antibody in 20 mM Tris-HCl at pH 8.0, at a volume of 2 µl / cm, and dry at 37 degrees overnight. S4: On the base liner 1 (size 80*300mm), the sample pad 4 (size 23*300mm, made of glass fiber cotton), the bonding pad 3 (size 10*300mm, made of glass fiber cotton), the coating film 2 (size 25*300mm, made of nitrocellulose) and the absorbent paper 5 (size 28*300mm) are sequentially overlapped to obtain the test paper, and then cut it into 4mm wide GDF-15 test cards as required.

[0052] The amount of GDF-15 monoclonal antibody 1 labeled with gold octahedral probe sprayed on the conjugate pad 2 is 5~20µg antibody / mL gold octahedral probe.

[0053] The detection line was coated with GDF-15 monoclonal antibody 2 (2 mg / ml), and the control line was coated with goat anti-mouse IgG antibody at a concentration of 2 mg / ml (the GDF-15 monoclonal antibody was purchased from Nanjing Baikang Biotechnology Co., Ltd., and the goat anti-mouse IgG antibody was from Changsha Boyou Biotechnology Co., Ltd.). The volume of coating solution used for the microsphere line was 2 µl / cm sample pad, and the volume of coating solution used for the detection line and control line was 0.5 µl / cm membrane.

[0054] The method for preparing the gold octahedral probe includes: A1: Synthesis of gold nanorods: In a container, 250 µL of 10 mM chloroauric acid was added to 9.75 mL of 0.1 M hexadecyltrimethylammonium bromide (CTAB) under magnetic stirring, followed by the addition of 0.6 mL of 10 mM sodium borohydride solution. After stirring for 2 min, the mixture was placed at 28 °C for 30 min. A2: Growth solution: 7.2 g CTAB and 0.987 g sodium oleate were dissolved in 400 mL of ultrapure water. Then, 20 mL of 10 mM chloroauric acid was injected into the solution. When the solution became colorless, 8 mL of 10 mM silver nitrate solution was added. After stirring for 5 min, 1.2 mL of hydrochloric acid and 600 µL of 0.1 M ascorbic acid were added. After reacting for about 30 s, 1 mL of seed solution was added. The reaction mixture was allowed to stand at 30 °C for 12 hours to complete the synthesis of gold nanorods. After synthesis, the nanorods were centrifuged at 9000 rpm for 15 min, washed twice, and dispersed in 20 mL of ultrapure water. A3: Synthesis of gold nano octahedrons: 50 mL of 10 mM CTAB and 500 µL of 0.05 M chloroauric acid were mixed and heated in a 60 °C water bath for 2 min. Then, 220 µL of 3-butenoic acid was added to the mixture. When the solution became colorless, 300 µL of gold nanorods were added and the mixture was allowed to stand in a 60 °C water bath for 12 hours. A4: Modification of Raman signal molecules: Take 5 mL of gold octahedral solution and add 250 μL of 10 -4The 4-mercaptobenzoic acid (4-MBA) solution of M was allowed to stand for 4 hours, then centrifuged to remove excess 4-MBA, and then resuspended in pure water. This completed the modification of the Raman signal molecule, resulting in AuNO@4-MBA.

[0055] A GDF-15 detection kit, wherein the detection card is prepared using the above-mentioned method for preparing GDF-15 detection cards.

[0056] The method of using the GDF-15 test kit is as follows: 80 μL of sample is applied to the sample pad. After 15 minutes, the sample is visually interpreted, and a portable Raman spectrometer with a 785 nm laser is used to acquire the Raman signal of the test strip's detection line with a laser power of 8.4 mW, an integration time of 5 s, and two cumulative counts. During the test, BWSpec4.0 software is used to baseline and smooth the raw data for noise reduction, and Origin software is used to perform curve fitting on the processed data.

[0057] (Example 6) The preparation method of a GDF-15 detection card in this embodiment includes the following steps: S1: Preparation of binding pad 3: Add GDF-15 monoclonal antibody 1 to the gold octahedral probe at 20 µg / mL, mix well, let stand for 10 min, add 10 µl / mL of 20% BSA for blocking, mix well, let stand for 5 min, centrifuge at 10000 rpm for 10 min, wash with 20 mM, pH 8.0 PBS buffer containing 1% BSA and resuspend to 20% of the original volume, and spray a line evenly on the binding pad with GDF-15 monoclonal antibody 1 labeled with gold octahedral probe diluted 6 times with microsphere dilution buffer containing 0.5% Tween-20, 2% BSA, 5% sucrose, 20 mM, pH 8.0 PBS buffer, using 4 µl liquid volume / cm sample pad, place in an oven and dry at 37°C overnight.

[0058] S2: Preparation of coating membrane 2: GDF-15 monoclonal antibody 2 and goat anti-mouse IgG antibody were used as detection lines and control lines, respectively, and were streaked parallel to each other on the nitrocellulose membrane for coating. The membrane was then dried. The concentrations of GDF-15 monoclonal antibody 2 and goat anti-mouse IgG antibody were adjusted to 0.5 mg / ml with 10 mM PB coating solution containing 2.5% trehalose. The amount of coating solution used was 1.5 µl / cm of membrane. The membrane was streaked parallel to each other on the nitrocellulose membrane as detection lines and control lines, respectively, with a 5 mm gap between the control lines and the detection lines. The membrane was then placed in an oven and dried at 45°C overnight. S3: Sample pad preparation 4: Spray two parallel lines evenly on one side of the conjugate pad with a sample pad treatment solution containing 0.5% NaCl, 0.5% S17, 1% BSA, 1 mg / ml anti-RBC antibody in 20mM Tris-HCl at pH 8.0. The amount of solution used is 2~4µl / cm. Dry at 37 degrees overnight. S4: On the base liner 1 (size 80*300mm), the sample pad 4 (size 23*300mm, made of glass fiber cotton), the bonding pad 3 (size 10*300mm, made of glass fiber cotton), the coating film 2 (size 25*300mm, made of nitrocellulose) and the absorbent paper 5 (size 28*300mm) are sequentially overlapped to obtain the test paper, and then cut it into 4mm wide GDF-15 test cards as required.

[0059] The sample pad 4 is coated with microspheres and the gold octahedral probe is labeled with GDF-15 monoclonal antibody 1. The coating membrane 2 is provided with a detection line 21 and a control line 22. The detection line 21 is close to the binding pad 3, and the detection line 21 and the control line 22 are parallel to each other and spaced 5 mm apart. The detection line 21 is coated with GDF-15 monoclonal antibody 2, and the control line 22 is coated with goat anti-mouse IgG antibody.

[0060] The amount of GDF-15 monoclonal antibody 1 labeled with gold octahedral probe sprayed on the conjugate pad 2 is 5~20µg antibody / mL gold octahedral probe.

[0061] The detection line was coated with GDF-15 monoclonal antibody 2 (0.5 mg / ml), and the control line was coated with goat anti-mouse IgG antibody at a concentration of 0.5 mg / ml (the GDF-15 monoclonal antibody was purchased from Nanjing Baikang Biotechnology Co., Ltd., and the goat anti-mouse IgG antibody was from Changsha Boyou Biotechnology Co., Ltd.). The volume of coating solution used for the microsphere line was 4 µl / cm sample pad, and the volume of coating solution used for the detection line and control line was 1.5 µl / cm membrane.

[0062] The method for preparing the gold octahedral probe includes: A1: Synthesis of gold nanorods: In a container, 250 µL of 10 mM chloroauric acid was added to 9.75 mL of 0.1 M hexadecyltrimethylammonium bromide (CTAB) under magnetic stirring, followed by the addition of 0.6 mL of 10 mM sodium borohydride solution. After stirring for 2 min, the mixture was placed at 28 °C for 30 min. A2: Growth solution: 7.2 g CTAB and 0.987 g sodium oleate were dissolved in 400 mL of ultrapure water. Then, 20 mL of 10 mM chloroauric acid was injected into the solution. When the solution became colorless, 8 mL of 10 mM silver nitrate solution was added. After stirring for 5 min, 1.2 mL of hydrochloric acid and 600 µL of 0.1 M ascorbic acid were added. After reacting for about 30 s, 1 mL of seed solution was added. The reaction mixture was allowed to stand at 30 °C for 12 hours to complete the synthesis of gold nanorods. After synthesis, the nanorods were centrifuged at 9000 rpm for 15 min, washed twice, and dispersed in 20 mL of ultrapure water. A3: Synthesis of gold nano octahedrons: 50 mL of 10 mM CTAB and 500 µL of 0.05 M chloroauric acid were mixed and heated in a 60 °C water bath for 2 min. Then, 220 µL of 3-butenoic acid was added to the mixture. When the solution became colorless, 300 µL of gold nanorods were added and the mixture was allowed to stand in a 60 °C water bath for 12 hours. A4: Modification of Raman signal molecules: Take 5 mL of gold octahedral solution and add 250 μL of 10 -4 The 4-mercaptobenzoic acid (4-MBA) solution of M was allowed to stand for 4 hours, then centrifuged to remove excess 4-MBA, and then resuspended in pure water. This completed the modification of the Raman signal molecule, resulting in AuNO@4-MBA.

[0063] A GDF-15 detection kit, wherein the detection card is prepared using the above-mentioned method for preparing GDF-15 detection cards.

[0064] The method of using the GDF-15 test kit is as follows: 80 μL of sample is applied to the sample pad. After 15 minutes, the sample is visually interpreted, and a portable Raman spectrometer with a 785 nm laser is used to acquire the Raman signal of the test strip's detection line with a laser power of 8.4 mW, an integration time of 5 s, and two cumulative counts. During the test, BWSpec4.0 software is used to baseline and smooth the raw data for noise reduction, and Origin software is used to perform curve fitting on the processed data.

[0065] Among them, the SEM of the sample prepared in Example 1 is as follows: Figure 1 As shown, the structure of the prepared GDF-15 detection card is as follows. Figure 2 As shown.

[0066] Using the samples prepared in Example 1, the antigen calibrator was diluted with negative clinical samples to obtain concentrations of 30000, 20000, 12000, 6000, 1200, 600, and 25 pg / mL. Each sample was measured three times, and the average value of the T-line Raman characteristic peak was used to establish a standard curve with the calibrator concentration. The standard curve was plotted using the antigen concentration and the average value of the T-line Raman characteristic peak of the sample signal. The curve data are shown in Table 1.

[0067] Standard curve such as Figure 3 As shown in the figure. The R value for GDF-15 is 0.9985, and this mark is used to quantitatively determine the concentration of GDF-15 in the sample. The performance of the GDF-15 detection card is then tested: (1) Limit of detection: The zero-value sample was repeatedly measured 20 times, and the mean M and standard deviation SD of the 20 results were calculated. The limit of detection of the method (M+2SD) was reported as the blank mean plus twice the standard deviation. The result of GDF-15 was 21.54 pg / mL, which meets the sensitivity standard of 25 pg / mL.

[0068] (2) Linear range: Seven concentration values ​​of GDF-15 from 25 to 30000 pg / mL were taken respectively, and each concentration was measured three times. The average value of the measured concentration was compared with the theoretical concentration for linear analysis. The linear equation of GDF-15 was obtained as y = 0.9244x + 382.29, r = 0.9970, which shows that the GDF-15 detection kit of the present invention has good correlation within the linear range.

[0069] (3) Precision: Three batches of the kits in this embodiment were taken, and the intra-batch CV of the repeatability control samples were tested. Each batch of kits was tested 10 times in parallel with the repeatability control samples. The intra-batch CVs of the three batches of GDF-15 120000pg / mL were 9.29%, 7.58%, and 6.28%, respectively, and the inter-batch CV was 9.48%. The intra-batch CVs of the three batches of 1200pg / mL were 7.16%, 6.22%, and 7.95%, respectively, and the inter-batch CV was 9.77%, all within 10%.

[0070] (4) Accuracy: The quality control material of the baseline sample was selected as the test sample and divided into three equal volumes. Different concentrations of accuracy control material were added to two of the samples to prepare two recovery samples with different added concentrations. The concentration of the added analyte was calculated. The same amount of solution without the analyte was added to the other sample to prepare the baseline sample. The samples were analyzed three times repeatedly, and the average value was used for calculation. The recovery rate was calculated as (analyte concentration - baseline sample concentration) / added concentration × 100%. The recovery rate of the GDF-15 recovered sample at 20000 pg / mL was 96.22%, the recovery rate at 12000 pg / mL was 91.49%, and the average recovery rate was 93.86%. The deviation was within 10%.

[0071] Continuous observation of the sample in Example 1 revealed the following color change in the T-line: Figure 5 The concentrations from left to right are 30000, 20000, 12000, 6000, 1200, 600, 25, and 0 pg / mL.

[0072] One hundred blood samples each for GDF-15 testing were collected from hospitals. The kits described in Examples 1-6 of this invention and the direct chemiluminescence immunoassay kit for growth differentiation factor-15 from Mindray Bio-Medical Electronics Co., Ltd. were used for comparative analysis. In the kit of this invention, 80 µl of blood sample was added to the sample well of the test card, and after chromatography for 10 min, the concentration was read using a Raman spectrometer. The same sample was compared using the direct chemiluminescence immunoassay kit for growth differentiation factor-15 from Mindray Bio-Medical Electronics Co., Ltd. Linearity analysis was performed on the test results, such as... Figure 4 As shown, the correlation is very good, GDF-15 r=0.9850, P>0.05, and the average relative deviation is less than 10%. The results meet the requirements of clinical analysis and are suitable for clinical testing.

[0073] As can be seen, the kit of the present invention utilizes the sensitivity of rare earth nano-fluorescence immunochromatography technology, combined with a dry immunofluorescence analyzer, to achieve highly sensitive, accurate, simple and rapid quantitative detection of GDF-15 antibody.

[0074] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a GDF-15 test kit, characterized by: Includes the following steps: S1: Preparation of binding pad: Add gold octahedral probe to GDF-15 monoclonal antibody 1, block with BSA, centrifuge at high speed, wash and resuspend with preservation solution, spray microspheres on the binding pad with GDF-15 monoclonal antibody 1 solution labeled with gold octahedral probe, and dry overnight. S2: Preparation of coating membrane: GDF-15 monoclonal antibody 2 and goat anti-mouse IgG antibody were used as the detection line and control line, respectively, and were stretched parallel to each other on the nitrocellulose membrane for coating. The membrane was then dried overnight. S3: Sample pad preparation: Treat the sample pad with sample pad treatment solution and dry overnight; S4: On the substrate, the sample pad, bonding pad, coating film and absorbent paper are sequentially pasted on each other to obtain the test paper, which is then cut to obtain the GDF-15 test card; The composition of the gold octahedral probe in step S1 is AuNO@4-MBA, where AuNO is a gold octahedron and @ indicates that the Raman signal molecule 4-mercaptobenzoic acid is modified on the surface of AuNO. The sample pad treatment solution in step S3 consists of 0.5% NaCl, 0.5% S17, 1% BSA, 1 mg / ml anti-RBC antibody, and 20 mM pH 8.0 Tris-HCl.

2. The method for preparing a GDF-15 detection card according to claim 1, characterized in that: In step S1, the amount of GDF-15 monoclonal antibody 1 added is 5~20µg / mL, and the BSA concentration is 20%.

3. The method for preparing a GDF-15 detection card according to claim 1, characterized in that: In the detection line, the GDF-15 monoclonal antibody 2 coating concentration is 0.1~2mg / ml, and the volume is 0.5~1.5µl of coating solution / cm membrane. In the quality control line, the goat anti-mouse IgG antibody coating concentration is 0.5~2mg / ml, and the volume is 0.5~1.5µl of coating solution / cm membrane.

4. The method for preparing a GDF-15 detection card according to claim 1, characterized in that: Add the gold octahedral probe to GDF-15 monoclonal antibody 1, block with BSA, centrifuge at high speed, wash and resuspend with preservation solution, and then dilute 4-10 times with microsphere diluent to obtain a solution of gold octahedral probe-labeled GDF-15 monoclonal antibody 1. The microsphere diluent consists of 0.5% Tween-20, 2% BSA, 5% sucrose, and 20mM, pH 8.0 PBS.

5. A method for preparing a GDF-15 detection card according to any one of claims 1 to 4, characterized in that: The method for preparing the gold octahedral probe includes: A1: Preparation of seed solution: Chloroauric acid was added to hexadecyltrimethylammonium bromide (CTAB) under magnetic stirring, sodium borohydride solution was added to dissolve, and the mixture was stirred and left to stand for 30 min; A2: Preparation of growth solution: CTAB and sodium oleate were dissolved in ultrapure water, chloroauric acid solution was added, and silver nitrate solution was added after the solution became colorless. After stirring, hydrochloric acid and ascorbic acid were added. After the reaction, seed solution was added. The reaction mixture was allowed to stand for 12 hours to complete the synthesis of gold nanorods. After centrifugation and washing, the nanorods were dispersed in ultrapure water. A3: Synthesis of gold nano octahedrons: CTAB and chloroauric acid were mixed and heated in a 60°C water bath. 3-Butenoic acid was added. After the reaction became colorless, gold nanorods were added and the mixture was allowed to stand in a 60°C water bath for 12 hours to obtain a gold octahedron solution. A4: Modification of Raman signal molecules: Add 4-mercaptobenzoic acid (4-MBA) solution to the gold octahedral solution, let stand for 4 hours, centrifuge to remove excess 4-MBA, and resuspend in pure water.

6. A GDF-15 detection kit, wherein the detection card is prepared using the preparation method of a GDF-15 detection card as described in any one of claims 1 to 5.

7. The method of using the GDF-15 detection kit for non-diagnostic purposes according to claim 6, characterized in that: Take 80 μL of sample and place it on the sample pad. After 15 minutes, visually interpret the result and use a portable Raman spectrometer with a 785 nm laser to collect the Raman signal of the test strip detection line with a laser power of 8.4 mW, an integration time of 5 s, and two cumulative counts. During the testing process, BWSpec4.0 software was used to baseline and smooth the original data for noise reduction, and Origin software was used to perform curve fitting on the processed data.

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