Diquat and paraquat colloidal gold immunochromatography joint detection card, preparation method and application

Abstract

This invention discloses a colloidal gold immunochromatographic assay card for rapid on-site detection of paraquat and diquat, its preparation method, and its application. The assay card includes a PCV substrate, with a sample pad, a gold-labeled pad, a nitrocellulose membrane, and an absorbent pad arranged sequentially along the chromatography direction. The gold-labeled pad adsorbs paraquat colloidal gold-labeled antibody solution and diquat colloidal gold-labeled antibody solution. The nitrocellulose membrane has a T1 detection line, a T2 detection line, and a control line along the chromatography direction. The T1 detection line area is coated with paraquat complete antigen, the T2 detection line area is coated with diquat complete antigen, and the control line is coated with goat anti-mouse IgG. The assay card has the advantages of good stability, high sensitivity, and high specificity. It can simultaneously detect diquat and paraquat under different matrices. Diquat and paraquat react independently during detection, which can avoid false positives and prevent improper treatment methods from affecting the success rate of rescue.

Description

Technical Field

[0001] This invention belongs to the field of rapid on-site detection, specifically the colloidal gold immunochromatographic assay card for diquat and paraquat and its application. Background Technology

[0002] Both diquat and paraquat belong to the bipyridine class of herbicides and have similar compositions. However, diquat is less toxic than paraquat. The lethal dose of diquat for adults is 6–12 g, with a mortality rate of 20–60%. Its damaging effect on the body is inversely related to the dose. The lethal dose of paraquat for adults is 5–15 ml of a 20% aqueous solution (20–40 mg / kg), with a mortality rate as high as 90%. The success rate of rescue is low. Due to its danger and the lack of an antidote or specific treatment, my country has banned its production, sale, and use since July 2016. With the ban on paraquat in my country, diquat, as a substitute for paraquat, has been increasingly used in agricultural production.

[0003] Although diquat is less toxic than paraquat, its weeding effect is lower and its price is higher. Some unscrupulous merchants mix paraquat with diquat or use counterfeit products. Paraquat and diquat have similar structures and physicochemical properties, but their toxicity and target organs are different. Paraquat targets the lungs, causing pulmonary fibrosis, which seriously affects respiratory function and is a major cause of death. Diquat, on the other hand, mainly causes damage to the kidneys, heart, and central nervous system. If a mixed herbicide of diquat and paraquat is accidentally ingested, or if paraquat is used as a counterfeit diquat herbicide, it is necessary to get tested in time and take appropriate rescue measures.

[0004] Existing methods for detecting paraquat and diquat mainly include gas chromatography, mass spectrometry, high-performance liquid chromatography, ultraviolet spectrophotometry, and capillary electrophoresis. While these methods can quantitatively or qualitatively detect paraquat and diquat with good specificity, they suffer from drawbacks such as cumbersome testing processes, long testing times, high dependence on testing equipment and related professionals, and high costs, making them unsuitable for widespread clinical application.

[0005] For example, patent CN111781199A discloses a method for rapid detection of paraquat and diquat in urine, including step 1, preparing urine solutions containing paraquat and diquat at different concentrations; step 2, making a colorimetric table; step 3, preparing test tubes; step 4, preparing reagents; step 5, combining the urine solution to be tested with the reagents; and step 6, observing the color change of the urine solution.

[0006] This method utilizes the principle that paraquat and diquat contain conjugated structures and have a pH value above 10, forming free ions and producing a blue (paraquat) or green (diquat) color, for the detection of paraquat or diquat. However, this method is cumbersome and has low sensitivity. Furthermore, when paraquat and diquat are present in urine at similar concentrations, their similar color development can easily interfere with each other, affecting the detection results and potentially leading to misuse of resuscitation methods and a lower success rate. Therefore, we propose a colloidal gold immunochromatographic assay card for diquat and paraquat and its application. Summary of the Invention

[0007] The purpose of this invention is to provide a colloidal gold immunochromatographic assay card for diquat and paraquat, its preparation method, and its application, enabling the simultaneous and rapid detection of two toxic substances, diquat and paraquat. It also addresses the problem in existing technologies where, when the concentrations of paraquat and diquat are similar, their color development is similar, easily causing interference and affecting the detection results.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows: a paraquat and diquat colloidal gold immunochromatographic assay card, comprising a PCV substrate, wherein the PCV substrate is provided with a sample pad, a gold-labeled pad, a nitrocellulose membrane and an absorbent pad in sequence along the chromatography direction; the gold-labeled pad adsorbs paraquat colloidal gold-labeled antibody solution and diquat colloidal gold-labeled antibody solution; the nitrocellulose membrane is provided with a T1 detection line, a T2 detection line and a control line along the chromatography direction; the T1 detection line region is coated with paraquat complete antigen, the T2 detection line region is coated with diquat complete antigen, and the control line is coated with goat anti-mouse IgG.

[0009] The principles and advantages of this solution are as follows:

[0010] Because it uses a joint detection card, it has the advantage of rapid detection. The joint detection card is coated with both paraquat complete antigen and diquat complete antigen, which can be detected at the same time, thus realizing the simultaneous and rapid detection of paraquat and diquat.

[0011] A sample solution containing any one of paraquat, diquat, or a mixture of both is dropped into the sample pad. When the sample solution passes through the gold-labeled pad containing colloidal gold-labeled antibody solutions of paraquat and diquat, the paraquat component reacts with the paraquat colloidal gold-labeled antibody solution, and the diquat component reacts with the diquat colloidal gold-labeled antibody solution. The reactions of paraquat and diquat with the colloidal gold-labeled antibody solutions do not interfere with each other. When passing through the T1 and T2 detection lines, they can bind to the complete antigens of paraquat and diquat, respectively. Therefore, even if the concentrations of paraquat and diquat are similar, their binding regions and color development regions are different, and the detection results will not interfere with each other.

[0012] In existing technologies, when paraquat and diquat concentrations are similar, their colors are similar, which can easily interfere with each other and affect the test results. However, in this method, diquat and paraquat are not interfered with each other during detection, and the results can be accurately identified, avoiding false positives that could lead to misuse of treatment plans and affect the success rate of rescue. Therefore, this method has higher sensitivity than existing technologies.

[0013] In addition, this method eliminates the need for sample pretreatment during testing, which not only improves detection efficiency but also avoids the significant impact of the mobile phase and pretreatment solution on the detection of diquat due to its high polarity, which can easily lead to double peaks in the chromatogram and affect the accuracy of the test results when using liquid chromatography. Experimental tests show that the detection speed of the combined test card in this method is 5-10 minutes, the sensitivity is 20 ng / ml, and the detection accuracy reaches 97%. Compared with large instruments such as liquid chromatography-mass spectrometry (LC-MS), the sensitivity is comparable and the detection result error is small, but the detection speed is faster than LC-MS. It can provide diagnostic evidence for paraquat and diquat poisoning in the first instance, gain valuable rescue time, improve the survival rate of poisoned patients, and is suitable for rapid detection and batch screening at the poisoning site and in the laboratory.

[0014] Furthermore, the nitrocellulose membrane has three channels along its length, with the bottom of the channels penetrating the nitrocellulose membrane and extending into the PVC base plate. A first air bladder is adhered to the lower side wall of the channel, and an insulation layer is adhered to the middle of the side wall of the channel. Frames are slidably fitted to the side walls of the channels. The three frames are respectively filled with paraquat complete antigen, diquat complete antigen, and goat anti-mouse IgG. The frame filled with paraquat complete antigen forms the T1 detection line, the frame filled with diquat complete antigen forms the T2 detection line, and the frame filled with goat anti-mouse IgG forms the quality control line. A buffer groove is opened on the top of the PVC base plate near the sample pad. A second air bladder and a PAM absorbent membrane are adhered to the buffer groove. The first air bladder is connected to the second air bladder. In the initial state, the frame is located in the middle of the side wall of the channel, and the first and second air bladders are inflated.

[0015] Beneficial effects:

[0016] 1. This method incorporates a buffer tank on the PCV base plate. When the sample solution is dripped into the sample pad, a portion of the solution is temporarily stored in the buffer tank and the PAM absorbent membrane, slowing down the flow of the solution towards the gold-labeled pad. This prevents the sample solution from flowing too quickly, which could prevent paraquat or diquat components from binding to the complete paraquat antigen or diquat antigen in time, thus affecting the accuracy of the detection results. Furthermore, the PAM absorbent membrane has excellent water absorption capacity. When a low-concentration sample solution is temporarily stored in the buffer tank, the water in the sample solution can be absorbed by the PAM absorbent membrane, increasing the concentration of the sample solution flowing towards the gold-labeled pad. This allows the paraquat component in the sample solution to bind to the complete paraquat antigen or the diquat component to bind to the complete diquat antigen more accurately. Therefore, even if the concentration of paraquat or diquat is low before the sample solution is dripped in, it can still be detected after being concentrated by the PAM absorbent membrane. This results in high sensitivity of the test card in this method.

[0017] 2. Before using the test card, embed the T1 test line, T2 test line and quality control line in the through groove. In this solution, the insulation layer can be a cold storage material with a phase change temperature of -20℃ to 0℃, so that the paraquat complete antigen, diquat complete antigen and sheep anti-mouse IgG are kept warm under the action of the insulation layer, maintaining their activity and function, and extending the shelf life of the test card.

[0018] 3. A second air bladder is bonded between the buffer tank and the PAM absorbent membrane. This serves two purposes: first, it prevents sample leakage along the buffer tank; second, when sample accumulates in the buffer tank, the weight of the sample compresses the second air bladder, causing the gas in the second air bladder to flow towards the first air bladder. This inflates the first air bladder, pushing the frame upwards and exposing the T1, T2, and control lines to the nitrocellulose membrane surface. This allows the sample passing through the gold-labeled pad to bind with paraquat complete antigen, diquat complete antigen, and goat anti-mouse IgG, detecting the presence of paraquat or diquat in the sample. The weight of the sample changes the flow of gas in the first and second air bladders, controlling the frame's position so that the T1, T2, and control lines are only exposed when using the test card. This extends the test card's shelf life and prevents contamination of the T1, T2, and control lines, thus avoiding interference with the test results.

[0019] Furthermore, the preparation method of the combined immunochromatographic test card for diquat and paraquat is as follows:

[0020] Step a: Preparation of colloidal gold solution: Colloidal gold solution is prepared using chloroauric acid and sodium citrate as raw materials;

[0021] Step b: Preparation of paraquat and diquat colloidal gold-labeled antibody solutions: Adjust the pH of the colloidal gold solution prepared in step a to 8.0-8.5 with potassium carbonate solution. Add paraquat and diquat monoclonal antibodies to the colloidal gold solution according to the specified amounts and stir for 1 hour. Add bovine serum albumin dropwise and stir for 1 hour to obtain a labeled colloidal gold solution with a volume concentration of 0.9-1.1%. Centrifuge the labeled colloidal gold solution at 9500-10500 r / min for 15 minutes, discard the supernatant, and resuspend the precipitate with resuspension to obtain paraquat colloidal gold-labeled antibody solution and diquat colloidal gold-labeled antibody solution, respectively. Store at 4℃ for later use.

[0022] Step c: Prepare the gold label pad;

[0023] Step d: Coating the T1 detection line, T2 detection line, and control line onto the nitrocellulose membrane: Paraquat complete antigen, diquat complete antigen, and goat anti-mouse IgG were coated onto the T1 detection line, T2 detection line, and control line regions, respectively.

[0024] Step e: Assemble the paraquat and diquat colloidal gold immunochromatographic assay card.

[0025] Further, in step a, the glassware is rinsed with aqua regia before preparation. 99 mL of distilled water and 1 mL of 1% chloroauric acid are added to an oil bath at a constant temperature of 108°C for preheating. When the reflux water flows to the three-necked flask, 4 mL of 1% sodium citrate is added and the mixture is stirred and heated for 30 min until the solution turns from purple to red. The solution is then cooled and stirred to room temperature to obtain a colloidal gold solution containing colloidal gold particles with a particle size of 10-30 nm.

[0026] Further, in step b, the resuspension is a mixture of equal volumes of 0.02-0.1 mol / L Tris-HCl, 3%-7% bovine serum albumin, 0.1%-0.3% Triton X-100, and 8%-12% sucrose.

[0027] Further, in step c, the paraquat colloidal gold-labeled antibody solution and the diquat colloidal gold-labeled antibody solution prepared in step b are mixed evenly in proportion, the gold-labeled pad is immersed in the mixed solution, and then dried at 35-40℃ for later use.

[0028] Further, in step d, the concentration of paraquat complete antigen is 0.2-1.0 mg / ml, the concentration of diquat complete antigen is 0.2-1.0 mg / ml, the concentration of goat anti-mouse IgG is 0.5-1.5 mg / ml, and the coated nitrocellulose membrane is dried at 35-40℃ for later use.

[0029] Further, in step e, the sample pad, gold label pad, coated nitrocellulose membrane and absorbent pad are sequentially bonded to the PCV substrate, cut into strips, and a combined detection card for paraquat and diquat colloidal gold immunochromatographic assay is prepared.

[0030] The preparation method proposed in this scheme has the following beneficial effects:

[0031] 1. Preheating chloroauric acid: On the one hand, it can increase the reaction rate, and on the other hand, it can promote solubility, which facilitates the full reaction with sodium citrate to obtain colloidal gold particles with uniform particle size;

[0032] 2. Potassium carbonate is a relatively mild pH adjuster capable of regulating a wide range of pH values. It does not strongly corrode colloidal gold solutions, and adjusting the pH with potassium carbonate solution will not affect the quality of the colloidal gold solution. However, since potassium carbonate can cause some colloidal gold particles to agglomerate, centrifugation can disperse them by collision, allowing them to fully bind with paraquat and diquat monoclonal antibodies. The Tris-HCl and Triton X-100 in the resuspension have good buffering effects, maintaining the pH value of the solution and better preserving the activity and function of paraquat and diquat monoclonal antibodies. The addition of bovine serum albumin can prevent non-specific binding, avoiding false positives caused by either paraquat or diquat components binding to paraquat antibodies when the prepared test strip is used for detection.

[0033] 3. Soaking the gold label pad in the mixed solution is to ensure that the paraquat and diquat components are in full contact with the mixed solution on the gold label pad when the prepared test card is used for testing, thus avoiding false positives.

[0034] Furthermore, the aforementioned paraquat and diquat colloidal gold immunochromatographic assay card can be applied to the detection of any type of specimen, including PBS solution, tap water, river water, urine, whole blood, plasma, and perfusion fluid.

[0035] Beneficial effects: The colloidal gold immunochromatographic assay card for diquat and paraquat proposed in this scheme can meet the needs of rapid detection of diquat and paraquat under various sample types.

[0036] Furthermore, the application method of the combined detection card for diquat and paraquat colloidal gold immunochromatographic assay is as follows:

[0037] S1. Take 70-100 μL of sample solution and drop it onto the colloidal gold immunochromatographic test card for diquat and paraquat, start timing, and read the results;

[0038] S2. Result Interpretation: The color development intensity of the T2 test line of the T1 test line is inversely correlated with the concentrations of paraquat and diquat.

[0039] (1) The T1 test line, T2 test line and quality control line all showed color: paraquat negative, diquat negative;

[0040] (2) The T1 test line is not colored, while the T2 test line and the quality control line are both colored: Paraquat positive, Diquat negative;

[0041] (3) The T2 test line is not colored, while the T1 test line and the quality control line are both colored: Paraquat negative, Diquat positive;

[0042] (4) The T1 and T2 test lines showed no color, while the control lines showed color: Paraquat positive and Diquat positive.

[0043] (5) The quality control line does not develop color: the joint inspection card test is invalid and needs to be retested.

[0044] Beneficial effects: By observing the different color development degrees of the T1 test line, T2 test line, and quality control line, the presence and concentration of paraquat and diquat can be more intuitively judged, providing a reliable basis for the diagnosis and rescue of paraquat and diquat poisoning. In addition, the joint detection card proposed in this scheme does not require sample pretreatment during detection, avoiding the large influence of diquat's high polarity, mobile phase, and pretreatment solution on the detection of diquat when using liquid chromatography, which can easily lead to double peaks in the spectrum and affect the accuracy of the detection results. Attached Figure Description

[0045] Figure 1 This is an isometric view of the paraquat and diquat colloidal gold immunochromatographic assay card of Example 1 of the present invention, wherein the arrow indicates the direction of chromatography.

[0046] Figure 2 This is a cross-sectional view of the paraquat and diquat colloidal gold immunochromatographic assay card of Example 2 of the present invention.

[0047] Figure 3 A schematic diagram illustrating the interpretation of results from the combined immunochromatographic assay of paraquat and glyphosate colloidal gold.

[0048] Figure 4 This diagram illustrates the results of a combined paraquat and diquat colloidal gold immunochromatographic assay card when different concentrations of paraquat and diquat are added to blood and urine. Detailed Implementation

[0049] The following detailed description illustrates the specific implementation method:

[0050] The reference numerals in the accompanying drawings include: PCV base plate 1, sample pad 2, gold label pad 3, nitrocellulose membrane 4, absorbent pad 5, T1 test line 6, T2 test line 7, quality control line 8, buffer tank 9, PAM absorbent membrane 10, second airbag 11, through groove 12, and first airbag 12.

[0051] Example 1:

[0052] The basic implementation examples are as follows: Figure 1 As shown:

[0053] The paraquat and diquat colloidal gold immunochromatographic assay card includes a PCV substrate 1. Along the chromatography direction, the PCV substrate 1 has a sample pad 2, a gold-labeled pad 3, a nitrocellulose membrane 4, and an absorbent pad 5. The gold-labeled pad 3 is adsorbed with paraquat colloidal gold-labeled antibody solution and diquat colloidal gold-labeled antibody solution. Along the chromatography direction, the nitrocellulose membrane 4 has a T1 detection line 6, a T2 detection line 6, and a control line 8. The T1 detection line 6 region is coated with paraquat complete antigen, the T2 detection line 6 region is coated with diquat complete antigen, and the control line 8 is coated with goat anti-mouse IgG.

[0054] Example 2:

[0055] The difference from Example 1 is that, as Figure 2 As shown, the nitrocellulose membrane 4 has three through-slots 12 along its length. The bottom of the through-slots 12 penetrates the nitrocellulose membrane 4 and extends into the PCV base plate 1. A first air bladder 12 is bonded to the lower side wall of the through-slot 12. An insulation layer is bonded to the middle of the side wall of the through-slot 12. Frames are slidably fitted to the side walls of the through-slots 12. The three frames are respectively filled with paraquat complete antigen, diquat complete antigen, and goat anti-mouse IgG. The frame filled with paraquat complete antigen forms the T1 detection line 6, the frame filled with diquat complete antigen forms the T2 detection line 7, and the frame filled with goat anti-mouse IgG forms the quality control line 8. A buffer groove 9 is opened on the top of the PCV base plate 1 near the sample pad 2. A second air bladder 11 and a PAM absorbent membrane 10 are bonded to the buffer groove 9. The first air bladder 12 is connected to the second air bladder 11. In the initial state, the frame is located in the middle of the side wall of the through-slot 12, and the first air bladder 12 and the second air bladder 11 are inflated.

[0056] Example 3:

[0057] The difference from Example 1 is that a method for preparing a colloidal gold immunochromatographic assay card for paraquat and diquat is also proposed, as follows:

[0058] Step a: Preparation of colloidal gold solution: Before preparation, glassware is rinsed with aqua regia. 99 mL of distilled water and 1 mL of 1% chloroauric acid are added to an oil bath at a constant temperature of 108℃ and preheated. When the reflux water flows to the three-necked flask, 4 mL of 1% sodium citrate is added and the mixture is stirred and heated for 30 min until the solution turns from purple to red. The solution is then cooled and stirred to room temperature to obtain a colloidal gold solution containing colloidal gold particles with a diameter of 20 nm.

[0059] Step b: Preparation of paraquat and diquat colloidal gold-labeled antibody solutions: Adjust the pH of the colloidal gold solution prepared in step a to 8.3 with potassium carbonate solution. Add paraquat and diquat monoclonal antibodies to the colloidal gold solution according to the specified amounts and stir for 1 hour. Add bovine serum albumin dropwise and stir for 1 hour to obtain a labeled colloidal gold solution with a volume concentration of 1.0%. Centrifuge the labeled colloidal gold solution at 10000 r / min for 15 minutes, discard the supernatant, and resuspend the precipitate with resuspension to obtain paraquat colloidal gold-labeled antibody solution and diquat colloidal gold-labeled antibody solution, respectively. Store at 4℃ for later use.

[0060] The resuspension was a mixture of equal volumes of 0.02-0.1 mol / L Tris-HCl, 5% bovine serum albumin, 0.2% Triton X-100, and 10% sucrose.

[0061] Step c: Preparation of gold-labeled pad 3: Mix the paraquat colloidal gold-labeled antibody solution and the diquat colloidal gold-labeled antibody solution prepared in step b at a ratio of 2:3. Soak the gold-labeled pad 3 in the mixed solution and dry it at 37°C for later use.

[0062] Step d: Coating T1 detection line 6, T2 detection line 7 and control line 8 onto nitrocellulose membrane 4: Paraquat complete antigen, diquat complete antigen and goat anti-mouse IgG are coated on the regions of T1 detection line 6, T2 detection line 7 and control line 8 respectively.

[0063] The concentrations of paraquat complete antigen and diquat complete antigen were 0.4 mg / ml, 0.6 mg / ml, and 1.0 mg / ml, respectively. The coated nitrocellulose membrane 4 was dried at 37°C for later use.

[0064] Step e: The sample pad 2, gold label pad 3, coated nitrocellulose membrane 4 and absorbent pad 5 are sequentially bonded to the PCV base plate 1, cut into strips, and the diquat and paraquat colloidal gold immunochromatographic test cards are prepared.

[0065] Example 4:

[0066] The difference from Example 1 is that, as shown in the appendix Figures 3-4 As shown, the application of the paraquat and diquat colloidal gold immunochromatographic assay card is also proposed. This card can be used to detect any type of specimen, including PBS solution, tap water, river water, urine, whole blood, plasma, and perfusion fluid. The application method is as follows:

[0067] S1. Take 70-100 μL of sample solution and drop it onto the colloidal gold immunochromatographic test card for diquat and paraquat, start timing, and read the results;

[0068] S2. Result Interpretation: The color development intensity of the T2 detection line 7 at the T1 detection line 6 is inversely correlated with the concentrations of paraquat and diquat.

[0069] (1) T1 test line 6, T2 test line 7 and quality control line 8 all showed color: paraquat negative, diquat negative;

[0070] (2) No color development was observed at T1 test line 6, while color development was observed at T2 test line 7 and control line 8: Paraquat positive, diquat negative;

[0071] (3) T2 test line 7 showed no color, while T1 test line 6 and quality control line 8 both showed color: paraquat negative, diquat positive;

[0072] (4) T1 test line 6 and T2 test line 7 showed no color, while control line 8 showed color: paraquat positive and diquat positive.

[0073] (5) No color development on quality control line 8: The joint inspection card test is invalid and needs to be retested.

[0074] The experimental data are as follows:

[0075] I. Sample preparation: Take 4000ml of urine and divide it into 4 equal parts. Add 20ug of paraquat to one part to prepare sample solution 1 with a concentration of 20ng / ml. Add 20ug of diquat to one part to prepare sample solution 2 with a concentration of 20ng / ml. Add 20ug of a mixture of paraquat and diquat to one part to prepare sample solution 3 with a concentration of 20ng / ml. Do not add paraquat or diquat to one part as sample solution 4.

[0076] II. Design a control group and an experimental group, and add sample solutions to each group respectively:

[0077] Take four test tubes and four sets of the prepared paraquat and diquat colloidal gold immunochromatographic assay cards. Add sample solution 1, sample solution 2, sample solution 3 and sample solution 4 to the test tubes respectively. After pretreatment, place them in a liquid chromatography instrument for detection. These serve as control group 1, control group 2, control group 3 and control group 4. Add sample solution 1, sample solution 2, sample solution 3 and sample solution 4 to the paraquat and diquat colloidal gold immunochromatographic assay cards respectively. Let them stand. These serve as experimental group 1, experimental group 2, experimental group 3 and experimental group 4.

[0078] III. Reading and recording results:

[0079] The results record includes the positive (negative) results for both the control and experimental groups, as well as the time of result issuance, as shown in Table 1:

[0080] Table 1 - Comparison of results for sample solutions 1, 2, 3, and 4 under liquid chromatography and combined detection card methods.

[0081] Sample liquid Yang (Yin) Time of issuance of results Experimental group 1 Paraquat positive, diquat negative 7min Experimental group 2 Paraquat is negative, diquat is positive. 8min Experimental group 3 Paraquat positive, diquat positive 10min Experimental group 4 Paraquat is negative, diquat is negative 5min Control group 1 Paraquat positive, diquat negative 5h Control group 2 Paraquat is negative, diquat is positive. 5h15min Control group 3 Paraquat positive, diquat positive 5h38min Control group 4 Paraquat is negative, diquat is negative 4h20min

[0082] As shown in Table 1, the results of the experimental group and the control group corresponded to the known additives in the sample solution. In this experiment, the detection accuracy of paraquat and diquat reached 100%, and the results of the experimental group were available faster than those of the control group.

[0083] This protocol also provides a comparison table of results for samples of different concentrations under liquid chromatography and combined detection cards for paraquat and diquat colloidal gold immunochromatography, as shown in Table 2:

[0084] Table 2 - Comparison of results of samples of different concentrations under liquid chromatography and combined detection cards for diquat and paraquat colloidal gold immunochromatography.

[0085]

[0086]

[0087] The validation results show that: 1. The kit has a fast detection speed, with a reaction time of only 5-10 minutes, far shorter than the 4-6 hours of traditional detection equipment; 2. It has high sensitivity, with a minimum detection concentration of 20 ng / mL, comparable to the detection limit of large-scale laboratory liquid chromatography-mass spectrometry (LC-MS); 3. It has strong specificity, with no cross-reaction when paraquat and diquat are detected simultaneously; 4. It is easy to operate, requiring no professional personnel or large equipment, and is suitable for rapid detection and batch screening at poisoning sites and in laboratories; 5. It is not affected by matrix interference and can be used for the detection of multiple sample types such as urine, plasma, and whole blood.

[0088] The development and use of this joint detection card will significantly enhance my country's ability to rapidly respond to paraquat and diquat poisoning emergencies, and provide a clinically practical rapid detection product for the clinical treatment, emergency response, and health risk screening of paraquat and diquat poisoning patients.

[0089] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0090] The above descriptions are merely embodiments of the present invention. Commonly known structures and characteristics are not described in detail here. Those skilled in the art are aware of all common technical knowledge in the field prior to the application date or priority date, are aware of all existing technologies in that field, and have the ability to apply conventional experimental methods prior to that date. Those skilled in the art can, under the guidance of this application, improve and implement this solution in combination with their own capabilities. Some typical known structures or methods should not be obstacles for those skilled in the art to implement this application. It should be noted that those skilled in the art can make several modifications and improvements without departing from the structure of the present invention. These should also be considered within the scope of protection of the present invention, and will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A combined immunochromatographic assay card for diquat and paraquat, characterized in that: The system includes a PCV substrate, which has a sample pad, a gold-labeled pad, a nitrocellulose membrane, and an absorbent pad arranged sequentially along the chromatography direction. The gold-labeled pad is adsorbed with paraquat colloidal gold-labeled antibody solution and diquat colloidal gold-labeled antibody solution. The nitrocellulose membrane has a T1 detection line, a T2 detection line, and a control line arranged along the chromatography direction. The T1 detection line area is coated with paraquat complete antigen, the T2 detection line area is coated with diquat complete antigen, and the control line is coated with goat anti-mouse IgG. The nitrocellulose membrane has three channels along its length, with the bottom of the channels penetrating the nitrocellulose membrane and extending into the PVC base plate. A first air bladder is adhered to the lower side wall of the channel, and an insulation layer is adhered to the middle of the side wall. Frames are slidably fitted to the side walls of the channels. The three frames are filled with paraquat complete antigen, diquat complete antigen, and goat anti-mouse IgG, respectively. The frame filled with paraquat complete antigen forms the T1 detection line, the frame filled with diquat complete antigen forms the T2 detection line, and the frame filled with goat anti-mouse IgG forms the control line. A buffer groove is opened on the top of the PVC base plate near the sample pad. A second air bladder and a PAM absorbent membrane are adhered to the buffer groove. The first air bladder is connected to the second air bladder. In the initial state, the frame is located in the middle of the side wall of the channel, and the first and second air bladders are inflated.

2. A method for preparing a colloidal gold immunochromatographic assay card for paraquat and diquat, characterized in that, Includes the following steps: Step a: Preparation of colloidal gold solution: Colloidal gold solution is prepared using chloroauric acid and sodium citrate as raw materials; Step b: Preparation of paraquat and diquat colloidal gold-labeled antibody solutions: Adjust the pH of the colloidal gold solution prepared in step a to 8.0-8.5 with potassium carbonate solution. Add the paraquat and diquat monoclonal antibodies to the colloidal gold solution according to the specified amounts and stir. Bovine serum albumin was added dropwise and stirred for 1 hour to prepare a labeled colloidal gold solution with a volume concentration of 0.9-1.1%. The labeled colloidal gold solution was centrifuged at 9500-10500 r / min for 15 min, the supernatant was discarded, and the precipitate was resuspended to prepare paraquat colloidal gold labeled antibody solution and diquat colloidal gold labeled antibody solution, which were stored at 4°C for later use. Step c: Prepare the gold label pad; Step d: Coating the T1 detection line, T2 detection line, and control line onto the nitrocellulose membrane: Paraquat complete antigen, diquat complete antigen, and goat anti-mouse IgG were coated onto the T1 detection line, T2 detection line, and control line regions, respectively. Step e: Assemble the paraquat and diquat colloidal gold immunochromatographic assay card.

3. The method for preparing the paraquat and diquat colloidal gold immunochromatographic assay card according to claim 2, characterized in that: In step a, the glassware was rinsed with aqua regia before preparation. 99 mL of distilled water and 1 mL of 1% chloroauric acid were added to an oil bath at 108°C for preheating. When the reflux water reached the three-necked flask, 4 mL of 1% sodium citrate was added, and stirring and heating continued for 30 minutes until the solution changed from purple to red. The solution was then cooled and stirred to room temperature to obtain a solution containing particles with a diameter of 10- Colloidal gold solution with 30nm colloidal gold particles.

4. The method for preparing the paraquat and diquat colloidal gold immunochromatographic assay card according to claim 2, characterized in that: In step b, the resuspension is a mixture of equal volumes of 0.02-0.1 mol / L Tris-HCl, 3%-7% bovine serum albumin, 0.1%-0.3% Triton X-100, and 8%-12% sucrose.

5. The method for preparing the paraquat and diquat colloidal gold immunochromatographic assay card according to claim 2, characterized in that: In step c, the paraquat colloidal gold-labeled antibody solution and the diquat colloidal gold-labeled antibody solution prepared in step b are mixed evenly in proportion. The gold-labeled pad is immersed in the mixed solution and dried at 35-40°C for later use.

6. The method for preparing the paraquat and diquat colloidal gold immunochromatographic assay card according to claim 2, characterized in that: In step d, the concentration of paraquat complete antigen is 0.2-1.0 mg / ml, and the concentration of diquat complete antigen is 0.2- The concentration of goat anti-mouse IgG was 0.5-1.5 mg / ml, and the coated nitrocellulose membrane was dried at 35-40°C for later use.

7. The method for preparing the paraquat and diquat colloidal gold immunochromatographic assay card according to claim 2, characterized in that: In step e, the sample pad, gold label pad, coated nitrocellulose membrane and absorbent pad are sequentially bonded to the PCV substrate, cut into strips, and a combined detection card for paraquat and diquat colloidal gold immunochromatography is prepared.

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