Iron-based porous carbon immunochromatographic test strip and its application

Abstract

The present invention relates to an iron-based porous carbon immunochromatographic test strip and its application. The iron-based porous carbon immunochromatographic test strip comprises a chromatography test strip, an OPD colorimetric reagent and an iron-based porous carbon-labeled monoclonal antibody reaction reagent for anti-identification of an object to be detected, wherein: the chromatography test strip comprises a cardboard, one side of the cardboard is sequentially pasted with a water-absorbing pad, a detection pad and a sample pad from top to bottom, and adjacent pads are overlapped and connected at the connection, the detection pad is based on a nitrocellulose membrane, and a horizontal quality control line and a detection line are arranged on the nitrocellulose membrane from top to bottom, the quality control line is coated with a sheep anti-mouse polyclonal antibody; the detection line is coated with an object to be detected-bovine serum albumin conjugate. It uses iron-based porous carbon as a labeling material, has a more obvious color development than nano-gold materials, is cheaper, and can catalyze color development, amplify the detection range and improve sensitivity. It is easy to operate, simple and fast.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and specifically relates to an iron-based porous carbon immunochromatographic test strip and application thereof. Background Art

[0002] As a promising portable rapid detection platform, test strip immunoassay technology has attracted widespread attention due to its advantages such as low cost, simple operation and instant analysis. At present, there are three main problems with traditional test strip detection technology: (1) insufficient sensitivity and high cost; (2) poor multiple detection and segmented detection capabilities and inability to quantify; (3) lack of portable user-friendly data analysis tools. Recently, artificial nanozymes have attracted widespread attention in the fields of biosensing, treatment and degradation of environmental pollutants. Due to their abundant raw material resources, excellent catalytic properties and good biocompatibility, iron-based nanozymes are used as the main material of beacon probes in biosensing analysis methods, but they are rarely reported in instant detection analysis technology. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide an iron-based porous carbon immunochromatographic test strip and its application. The test strip has the characteristics of high sensitivity, low cost, quantitative and adjustable.

[0004] The technical solution adopted by the present invention to solve the above technical problems is:

[0005] The invention discloses an iron-based porous carbon immunochromatographic test strip, comprising a chromatography test strip, an OPD colorimetric reagent and a monoclonal antibody reaction reagent for identifying an object to be detected and marked with iron-based porous carbon, wherein: the chromatography test strip comprises a cardboard, one side of the cardboard is sequentially pasted with a water-absorbing pad, a detection pad and a sample pad from top to bottom, and adjacent pads are overlapped and connected at the connection point; the detection pad uses a nitrocellulose membrane as a base pad, and a transverse quality control line and a detection line are arranged on the nitrocellulose membrane from top to bottom, and the quality control line is coated with a sheep anti-mouse polyclonal antibody; the detection line is coated with a to-be-detected object-bovine serum albumin conjugate, and the OPD (o-phenylenediamine) colorimetric reagent is a buffer solution with a pH of 3-5 containing OPD and hydrogen peroxide.

[0006] According to the above scheme, the concentration of hydrogen peroxide in the OPD color developing reagent is 0.3-0.4 mM; the concentration of OPD is 0.3-0.4 mM.

[0007] According to the above scheme, the absorbent pad of the iron-based porous carbon immunochromatographic test strip is 40-45 mm long and 3-5 mm wide, and is cut from absorbent paper; the detection pad is CN95, 22-28 mm long and 3-5 mm wide; the sample pad is FUSION 5, 12-16 mm long and 3-5 mm wide, and the overlapping length of adjacent pads is 1-3 mm; the distance between the detection line on the detection pad and the upper edge of the sample pad is 6-10 mm, and the distance between the quality control line and the detection line is 8-10 mm.

[0008] According to the above scheme, the coating amount of the object to be detected-bovine serum albumin conjugate required on the detection line of the test pad per centimeter width is 300-400ng; the coating amount of the goat anti-mouse polyclonal antibody required on the quality control line of the test strip per centimeter width is 120-160ng.

[0009] According to the above scheme, the analyte is fumonisin, and the IC50 of the anti-fumonisin monoclonal antibody is less than or equal to 0.32 ng / mL. Specifically, the anti-fumonisin monoclonal antibody secreted and produced by the hybridoma cell line Fm7A11 with a deposit number of CCTCC NO.C201636 can be selected.

[0010] According to the above scheme, the iron-based porous carbon has a particle size of 30-100 nm and a porous structure. The surface of the carbon particles has nanoclusters of iron and iron oxides with a nanometer size of less than 10 nm.

[0011] According to the above scheme, the iron-based porous carbon is prepared by in-situ high-temperature carbonization with zinc nitrate and 2-methylimidazole as precursors of the metal organic framework ZIF-8 and heme as the precursor of iron. The high-temperature carbonization temperature is 800-900°C and the carbonization time is 1-2h.

[0012] According to the above scheme, the mass percentage of hemoglobin is 1%-2% of zinc nitrate.

[0013] A method for preparing the above-mentioned iron-based porous carbon immunochromatographic test strip is provided, comprising the following steps:

[0014] Preparation of chromatography test strips:

[0015] (1) Preparation of detection pad

[0016] Test line coating:

[0017] The conjugate of the object to be detected and bovine serum albumin is prepared into a coating solution; it is coated horizontally on the nitrocellulose membrane by streaking at a position 6 to 10 mm from the upper edge of the sample pad to obtain a detection line, and then dried at 37° C. for 60 to 120 minutes;

[0018] Coating of quality control line:

[0019] Prepare a coating solution with sheep anti-mouse polyclonal antibody; coat it horizontally on a nitrocellulose membrane at a position 8 to 10 mm away from the detection line by streaking to obtain a quality control line, and then dry it at 37°C for 60 to 120 minutes;

[0020] (2) Assembly of test strips

[0021] On one side of the paperboard, a water-absorbing pad, a detection pad and a sample pad are sequentially pasted from top to bottom, and the adjacent pads are overlapped and connected at the joints, with the overlap length being 1 to 3 mm, to obtain a chromatography test strip;

[0022] An OPD colorimetric reagent is provided, and the preparation method is as follows: a hydrogen peroxide solution is added to the OPD reagent, and a buffer solution with a pH of 3-5 is added to obtain the OPD colorimetric reagent.

[0023] A monoclonal antibody for identifying an object to be detected and labeled with iron-based porous carbon is provided. The obtaining method is as follows: the iron-based porous carbon solution and the monoclonal antibody solution for identifying the object to be detected are mixed, and the mixture is stirred and centrifuged at room temperature.

[0024] Provided is an application method of the iron-based porous carbon immunochromatographic test strip: providing a sample liquid to be tested, adding a detection buffer, mixing, then taking a monoclonal antibody reaction reagent labeled with the iron-based porous carbon to identify the object to be detected, adding it thereto and mixing, inserting the immunochromatographic test strip, reacting, and after the black band of the quality control line appears, adding an OPD colorimetric reagent at the detection line, and after a period of time, measuring and obtaining the RGB value of the detection line, and performing quantitative analysis of the content of the object to be detected based on a standard curve of RGB value-concentration of the object to be detected.

[0025] According to the above scheme, the sample to be tested is an agricultural product such as corn or wheat, and the substance to be tested is fumonisin; the sample pretreatment method is: adding methanol aqueous solution to the sample to be tested, shaking and extracting, centrifuging to obtain the supernatant, and filtering to obtain the sample extract.

[0026] According to the above scheme, the volume ratio of the sample solution to be tested and the detection buffer is 1:2-3; the detection buffer is a PBS buffer solution containing 2% (v / v) tween-20, 0.1% (w / v) sucrose and 0.5% (w / v) BSA; the volume of the monoclonal antibody reaction reagent labeled with iron-based porous carbon that recognizes the object to be detected is 15-25% of the sample solution to be tested.

[0027] According to the above scheme, the quantitative equation of the RGB color value is: intensity = 0.3R + 0.59G + 0.11B, where R, G, and B are the color intensity values ​​of the red, yellow, and blue color of the detection line, respectively, which can be measured by a colorimeter or a camera.

[0028] According to the above scheme, the method for obtaining the standard curve is:

[0029] Prepare a series of standard solutions with gradient concentrations;

[0030] The above series of standard solutions with gradient concentrations were used as test solutions for detection. After the black band of the quality control line appeared, OPD colorimetric reagent was added for secondary detection to obtain the RGD value of the test line corresponding to each concentration of the standard solution. Based on the RGD value and the concentration of the standard solution, the RGB value-analyte concentration standard curve was obtained.

[0031] According to the above scheme, the analyte is fumonisin B1, and the concentration of fumonisin B1 in the preparation of the standard curve is 0.02-150 ng.mL -1 .

[0032] The present invention adopts iron-based porous carbon material as the marking material. The iron-based porous carbon has a particle size of 30-100nm and a porous structure. There are nanoclusters of iron and iron oxide on the surface of the carbon particles. The nanocluster size is less than 10nm. It has artificial peroxidase activity and is easy to bind to antibodies. It can bind to anti-fumonisin B1 monoclonal antibodies. The color development is more obvious than that of nanogold materials and the price is lower. Based on the H202 medium catalyzing the color development of OPD color developing reagent, it can expand the detection range and improve the sensitivity, and has great practical application value.

[0033] The beneficial effects of the present invention are:

[0034] (1) The test strip provided by the present invention has high detection sensitivity. There are two detection sections, and OPD can be added for secondary detection according to actual needs. The detection section can be adjusted, and the operation is simple and convenient; when OPD is added for secondary detection, the detection line changes from black or colorless to yellow, and the RGB value is obtained by the test. Further quantitative analysis is performed based on the RGB value, and the sensitivity is high.

[0035] (2) Rapid detection. The whole process only takes 10 minutes.

[0036] (3) Low detection cost. The iron-based porous carbon immunochromatographic test strip provided by the present invention uses an iron-based porous carbon material with artificial peroxidase activity and easy antibody binding as a labeling material. It has a more obvious color development than nano-gold materials and is cheaper. It can enlarge the detection range and improve the sensitivity, and has great practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is the SEM image of iron-based porous carbon;

[0038] Figure 2 TEM images of iron-based porous carbon (different magnifications);

[0039] Figure 3 BET diagram of iron-based porous carbon. DETAILED DESCRIPTION

[0040] Example 1: Preparation method of anti-fumonisin B1 monoclonal antibody reaction reagent labeled with iron-based porous carbon

[0041] The anti-fumonisin B1 monoclonal antibody is a monoclonal antibody secreted by the hybridoma cell line Fm7A11 with a deposit number of CCTCC NO.C201636, and is specifically prepared according to the method reported in the patent application number CN201710131166.0.

[0042] Preparation method of iron-based porous carbon:

[0043] Weigh 1.07g Zn(NO3)2·6H2O and dissolve it in 40mL methanol solution, mix quickly, quickly add another 40mL methanol containing 2.35g 2-methylimidazole and 10mg heme, stir vigorously at room temperature for 24h, collect the gray product by centrifugation, rinse with methanol 5 times, and then vacuum dry at 70℃ overnight. The obtained gray product powder is transferred to a porcelain boat and heated from room temperature to 900℃ at a heating rate of 5℃ / min under N2 conditions for 2h. After grinding, iron-based porous carbon with uniform particle size between 30-100nm is obtained.

[0044] SEM and TEM images of Fe-based porous carbon Figure 1 and 2 As shown, Figure 1 , Figure 2 It shows that the particle size is between 30-100nm, and nanoclusters of iron and iron oxide are attached to the particles, with a nanometer size of less than 10nm; the BET diagram is shown in Figure 3 As shown in the BET test results, the specific surface area of ​​the material is 890.0437m 2 / g, pore volume is 0.857657cm 3 / g, and the average pore diameter is 3.85445nm, indicating that the present invention is an iron-based porous carbon material.

[0045] Preparation method of anti-fumonisin B1 monoclonal antibody reaction reagent labeled with iron-based porous carbon:

[0046] Take 10 mg of iron-based porous carbon material and add 1 mL of 10 mM PBS buffer solution to mix well. Take 100 μL of the above solution and add 800 μL of 10 mM PBS buffer solution (pH = 7.4), mix well, and then add 100 μL of 0.25 mg / mL anti-FB1 monoclonal antibody PBS buffer solution dropwise. The mixed solution was gently stirred at room temperature for 4 hours, centrifuged at 4°C 10000 rpm for 20 min, and the resulting precipitate was re-dissolved with 1 mL of 10 mM PBS buffer solution containing 1% (m / v) BSA to obtain an iron-based porous carbon labeled anti-fumonisin B1 monoclonal antibody reaction reagent, which was placed at 4°C for use.

[0047] Example 2: Preparation of Fumonisin B1 Iron-based Porous Carbon Immunochromatographic Test Strips

[0048] (1) Preparation of absorbent pad

[0049] Cut the absorbent paper into the size of 45mm long and 3mm wide to obtain the absorbent pad;

[0050] (2) Preparation of detection pad

[0051] Paste the CN95 nitrocellulose membrane with a specification of 25mm in length and 3mm in width to the corresponding position of the cardboard and then spray the test line and quality control line respectively.

[0052] Test line coating:

[0053] Accurately weigh 2.9 g Na2HPO4·12H2O, 0.2 g KCl, 0.2 g KH2PO4 and 8.0 g NaCl, add ultrapure water to the volume with a 1 L volumetric flask, mix well, and prepare a PBS buffer solution.

[0054] Accurately weigh 1 mg of fumonisin B1-bovine serum albumin conjugate (FB1-BSA), prepare a coating solution with a concentration of 0.5 mg / mL with 2 mL of 10 mM PBS buffer solution, and laterally coat it on a nitrocellulose membrane by a film-scratching method at a speed of 0.6 μL / cm at a position 6 mm from the upper edge of the sample pad to obtain a detection line, and then dry it at 37° C. for 120 minutes. The coating amount of fumonisin B1-bovine serum albumin conjugate (FB1-BSA) required on the detection line of the test strip detection pad per centimeter width is 300 ng;

[0055] Coating of quality control line:

[0056] Accurately weigh 1 mg of goat anti-mouse polyclonal antibody and prepare it into a coating solution with a concentration of 0.2 mg / mL with 5 mL of 10 mM PBS buffer solution; the film-scraping speed is 0.6 μL / cm, and it is horizontally coated on the nitrocellulose membrane at a position 8 mm away from the detection line by film-scraping to obtain a quality control line. The coating amount of goat anti-mouse polyclonal antibody required for the quality control line of each centimeter width of the test strip is 120 ng, and then dried at 37°C for 120 minutes;

[0057] (3) Assembly of test strips

[0058] On one side of the cardboard, a water-absorbing pad, a detection pad and a sample pad are sequentially pasted from top to bottom, and the adjacent pads are overlapped and connected at the joints, with an overlap length of 2 mm, to obtain a chromatography test strip;

[0059] Preparation method of OPD colorimetric reagent: 100 μL of 10mM OPD reagent is added with 100 μL of 10mM hydrogen peroxide solution, and the volume is adjusted to 3 mL with 200 mM sodium acetate-acetic acid buffer solution with pH = 4.0; the 10mM OPD reagent is prepared by weighing 10.8 mg of OPD and dissolving it in 10 mL of DMF; the sodium acetate-acetic acid buffer solution (200 mM, pH = 4.0) contains 32.8 mg of sodium acetate and 120 μL of pure acetic acid per 50 mL of water.

[0060] Example 3: Application of fumonisin B1 iron-based porous carbon immunochromatographic test strips

[0061] 1. The relationship curve between the RGB value of the detection line of the fumonisin B1 iron-based porous carbon immunochromatographic test strip and the concentration of fumonisin B1 is established by intensity = 0.3R + 0.59G + 0.11B (where R, G, and B are the color intensity values ​​of the red, yellow, and blue color of the detection line, respectively):

[0062] (1) Sample pretreatment was performed on wheat samples that were negative for fumonisin by high performance liquid chromatography (HPLC). Weigh 5 g of wheat sample, add 15 mL of 70% methanol aqueous solution, mix well, vortex extract for 20 minutes, centrifuge at 4000 rpm for 10 minutes, take the supernatant, and filter through a 0.22 μm filter membrane to obtain;

[0063] (2) The above-mentioned grain sample dilutions were spiked with standard substances at concentrations of 0.02 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 2 ng / mL, 10 ng / mL, 50 ng / mL, 150 ng / mL, 200 ng / mL, and 250 ng / mL;

[0064] (3) Weigh 2 mL of tween-20, 0.1 g of sucrose and 0.5 g of BSA and dilute to 100 mL with PBS buffer to prepare the detection buffer.

[0065] (4) Weigh 10.8 mg of OPD and dissolve it in 10 mL of DMF to prepare 10 mM OPD reagent. Weigh 32.8 mg of sodium acetate and 120 μL of pure acetic acid, dilute with 50 mL of water to prepare sodium acetate-acetic acid buffer solution. Take 100 μL of 10 mM OPD reagent and add 100 μL of commercially available 30% hydrogen peroxide solution, dilute to 3 mL with 200 mM sodium acetate-acetic acid buffer solution with pH = 4.0 to prepare OPD colorimetric reagent.

[0066] (5) Take 50 μL of the standard solution and add it to 120 μL of the detection buffer, mix well, then take 10 μL of the iron-based porous carbon labeled anti-fumonisin B1 monoclonal antibody reaction reagent and add it, mix well, insert the immunochromatographic test strip, wait for 9 minutes, and after the black band of the quality control line appears, add 1 μL of OPD colorimetric reagent to the detection line. The detection line changes from black or colorless to yellow. After about 1 minute, take a photo of the detection line with a mobile phone to obtain the RGB value of the detection line band.

[0067] (6) The concentration of the fumonisin standard is used as the horizontal axis, and the intensity of the corresponding detection line of each concentration standard solution (intensity = 0.3R + 0.59G + 0.11B, where R, G, and B are the color intensity values ​​of the red, yellow, and blue color of the detection line, respectively) is used as the vertical axis to fit the relationship curve. The effective detection range of this method is 0.02-150 ng / mL of fumonisin.

[0068] 2. Spike recovery experiment of fumonisin content in wheat samples:

[0069] Take 5g of wheat sample that is negative for fumonisin by high performance liquid chromatography (HPLC), add 15mL of 70% methanol aqueous solution, mix, vortex extract for 20 minutes, centrifuge at 4000rmp for 10min, take the supernatant, and filter with 0.22μm filter membrane. Accurately add 0.1ng / mL, 0.5ng / mL and 0.8ng / mL of fumonisin standard respectively, take 50μL of the above-mentioned sample test solution and add 120μL of detection buffer, mix, then take 10μL of anti-fumonisin B1 monoclonal antibody reaction reagent labeled with iron-based porous carbon and add it, mix, insert immunochromatography test strip, wait for 9min of reaction, wait for the black band of the quality control line to appear, add 1μL of OPD colorimetric reagent at the detection line, the detection line changes from black or colorless to yellow, about 1min later, take a photo of the detection line with a mobile phone, and obtain the RGB value of the detection line band. The intensity value is calculated by the formula intensity = 0.3R + 0.59G + 0.11B, where R, G, and B are the color intensity values ​​of the red, yellow, and blue color of the detection line, respectively. The intensity value is substituted into the relationship curve between the detection line intensity of the fumonisin B1 iron-based porous carbon immunochromatographic test strip and the concentration of the standard substance obtained above to obtain the fumonisin content. The spiked recoveries of fumonisin in the samples were measured and analyzed to be 99.5%, 89.2%, and 106.1%, respectively.

Claims

1. An iron-based porous carbon immunochromatographic test strip, characterized in that: The invention comprises a chromatographic test strip, an OPD colorimetric reagent and an iron-based porous carbon-labeled monoclonal antibody reaction reagent for identifying an object to be detected, wherein: the chromatographic test strip comprises a cardboard, one side of which is sequentially pasted with a water-absorbing pad, a detection pad and a sample pad from top to bottom, and adjacent pads are overlapped and connected at the connection point; the detection pad is based on a nitrocellulose membrane, and a transverse quality control line and a detection line are arranged on the nitrocellulose membrane from top to bottom; the quality control line is coated with a sheep anti-mouse polyclonal antibody; the detection line is coated with a conjugate of an object to be detected-bovine serum albumin; the OPD colorimetric reagent is a PH value of 3 containing OPD and hydrogen peroxide. -5 buffer solution, the hydrogen peroxide concentration in the OPD colorimetric reagent is 0.3-0.4mM; the OPD concentration is 0.3-0.4mM, the iron-based porous carbon has a porous structure, a particle size of 30-100nm, and nanoclusters of iron and iron oxides are present on the surface of the carbon particles, with a nanometer size of less than 10nm. Zinc nitrate and 2-methylimidazole are used as precursors of the organic metal framework ZIF-8, and heme is used as a precursor of iron. It is prepared by in-situ high-temperature carbonization, the high-temperature carbonization temperature is 800-900°C, the carbonization time is 1-2h, and the heme is 1%-2% of the zinc nitrate by mass percentage.

2. The iron-based porous carbon immunochromatographic test strip according to claim 1, characterized in that: The absorbent pad of the chromatography test strip is 40 to 45 mm long and 3 to 5 mm wide, and is cut from absorbent paper; the detection pad is 22 to 28 mm long and 3 to 5 mm wide; the sample pad is 12 to 16 mm long and 3 to 5 mm wide, and the overlapping length of adjacent pads is 1 to 3 mm; the distance between the detection line on the detection pad and the upper edge of the sample pad is 6 to 10 mm, and the distance between the quality control line and the detection line is 8 to 10 mm.

3. The iron-based porous carbon immunochromatographic test strip according to claim 1, characterized in that: The coating amount of the object to be detected-bovine serum albumin conjugate required on the detection line of the test pad per centimeter width of the test strip is 300-400ng; the coating amount of the goat anti-mouse polyclonal antibody required on the quality control line of the test strip per centimeter width is 120-160ng.

4. The iron-based porous carbon immunochromatographic test strip according to claim 1, characterized in that: The object to be detected is fumonisin, and the monoclonal antibody for identifying the object to be detected is an anti-fumonisin monoclonal antibody with an IC50 of less than or equal to 0.32 ng / mL.

5. The method for preparing the iron-based porous carbon immunochromatographic test strip according to claim 1, characterized in that: The following steps are involved: Preparation of chromatography test strips: (1) Preparation of detection pad Test line coating: The conjugate of the object to be detected and bovine serum albumin is prepared into a coating solution; it is horizontally coated on the nitrocellulose membrane by streaking at a position 6 to 10 mm from the upper edge of the sample pad to obtain a detection line, and then dried at 37°C for 60 to 120 minutes; Coating of quality control line: Prepare a coating solution with sheep anti-mouse polyclonal antibody; coat it horizontally on a nitrocellulose membrane by streaking at a position 8 to 10 mm away from the detection line to obtain a quality control line, and then dry it at 37°C for 60 to 120 minutes; (2) Assembly of test strips On one side of the paperboard, a water-absorbing pad, a detection pad and a sample pad are sequentially pasted from top to bottom, and the adjacent pads are overlapped and connected at the joints, with an overlap length of 1 to 3 mm, to obtain a chromatography test strip; Provide OPD color development reagent; The invention provides a monoclonal antibody reaction reagent which is labeled with iron-based porous carbon and is capable of recognizing an object to be detected.

6. The use of the iron-based porous carbon immunochromatographic test strip according to claim 1, characterized in that: Application method: Provide a sample liquid to be tested, add a detection buffer, mix well, then take a reaction reagent of a monoclonal antibody that recognizes the object to be detected and labeled with iron-based porous carbon, add it thereto and mix well, insert the immunochromatographic test strip, react, and after the black band of the quality control line appears, add the OPD colorimetric reagent to the detection line. After a period of time, determine the RGB value of the detection line, and perform quantitative analysis of the content of the object to be detected based on the RGB value-concentration standard curve of the object to be detected.

7. The use according to claim 6, characterized in that: The sample to be tested is agricultural product, and the substance to be detected is fumonisin; The sample to be tested is added with methanol-water solution and shaken for extraction, and the supernatant obtained by centrifugation is filtered to obtain the sample solution to be tested; The detection buffer is a PBS buffer solution containing 2% (v / v) tween-20, 0.1% (w / v) sucrose and 0.5% (w / v) BSA; The quantitative equation of RGB value is: intensity = 0.3R + 0.59G + 0.11B, where R, G, and B are the color intensity values ​​of red, yellow, and blue of the detection line, respectively, which are measured by a colorimeter or a camera. The method for obtaining the RGB value-to-be-detected substance concentration standard curve is: Prepare a series of standard solutions with gradient concentrations; The above series of standard solutions with gradient concentrations were used as test solutions for detection. After the black band of the quality control line appeared, OPD was added for secondary detection to obtain the RGB value of the test line corresponding to each concentration of the standard solution. Based on the RGB value and the concentration of the standard solution, the RGB value-concentration standard curve of the object to be detected was obtained.

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