Immunochromatography test strip based on fluorescent microsphere / clastic enzyme double signals and application of immunochromatography test strip

Abstract

The invention discloses an immunochromatography test strip based on fluorescent microsphere / clastic enzyme double signals and application of the immunochromatography test strip, and relates to the field of immunochromatography detection. The immunochromatography test strip takes fluorescent microspheres / clastic enzyme as a signal label, the signal label is connected with an antibody (such as an anti-thiabendazole monoclonal antibody) of a substance to be detected to serve as a signal probe, quantitative detection is performed by utilizing fluorescence characteristics of the fluorescent microspheres, and compared with traditional colorimetry, the immunochromatography test strip has a wider detection range and a lower detection limit; h2O2 is catalyzed by a clastic enzyme to generate O2, and the thiabendazole in a sample is detected by the double-signal immunochromatography test strip according to the generation amount of oxygen and a fluorescence value. Compared with a traditional colloidal gold immunochromatography test strip, the colloidal gold immunochromatography test strip disclosed by the invention is stronger in color development, wider in detection range, higher in sensitivity, better in stability, capable of realizing qualitative and quantitative detection of a sample, simple and convenient to operate and suitable for on-site rapid detection, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of immunochromatographic detection, and in particular to an immunochromatographic test strip based on fluorescent microspheres / degrading enzyme dual signals and applications thereof. Background Art

[0002] Thiabendazole is a benzimidazole pesticide. It is an antifungal and anthelmintic approved by the US Food and Drug Administration (FDA). Pre- and post-harvest treatment of fruits and vegetables with TBZ can reduce mold, pests, and rot, as well as deterioration during storage and transportation. TBZ is one of the most frequently tested pesticides in Europe and the United States. It is known that it cannot be effectively removed from fruit by washing and is stable during food processing. The European Food Safety Authority (EFSA) allows maximum residue levels of TBZ of 10 mg / kg for papaya, 7 mg / kg for citrus fruits and mangoes, 6 mg / kg for bananas, 4 mg / kg for pears, 3 mg / kg for sweet potatoes, 0.05 mg / kg for tea and coffee beans, less than 0.02 mg / kg for other fruits and vegetables, and less than mg / kg for animal-derived products. Although TBZ has low toxicity, with a toxicity category of 4 (the least toxic), the US Environmental Protection Agency (EPA) classifies it as possibly carcinogenic, and doses high enough to cause thyroid hormone imbalance. According to the US Environmental Protection Agency (EPA), the Acceptable Daily Intake (ADI) for TBZ is 0.1 mg / kg, while the World Health Organization (WHO) stipulates it is 0.3 mg / kg. For single-dose exposure, the No Observed Effect Level (NOEL) is 3.3 mg / kg.

[0003] Currently, the four main detection technologies for thiabendazole include chromatography, spectroscopy, electrochemical sensors, and immunochromatographic strips. Chromatographic methods (such as HPLC and LC-MS / MS) can simultaneously detect multiple benzimidazoles due to their high sensitivity and strong specificity. However, they require expensive instrumentation, complex pretreatment, and specialized personnel, resulting in high costs and time-consuming procedures, making them difficult to meet the needs of rapid on-site testing. UV-visible spectrophotometry, while simple to operate and low-cost, has low sensitivity and is susceptible to interference from the sample matrix, making it suitable only for preliminary screening. Electrochemical sensors achieve rapid response through molecularly imprinted or nanomaterial-modified electrodes, offering the advantages of portability. However, their long-term stability and reproducibility still need to be improved, and the technology is not readily adaptable to grassroots units.

[0004] In the field of rapid detection, immunochromatographic strips have become a hot topic in environmental monitoring, food testing, and medical diagnosis due to their simplicity (no professional training required), rapid response time (5-15 minutes), low cost, and portability. To improve detection sensitivity, novel fluorescent microspheres are used as signal labels. Compared to traditional colorimetric methods, these are more stable, have lower detection limits, and offer a wider detection range. Nanozymes are used as signal amplifiers, enabling rapid dual-signal detection based on the generated O2 content, significantly improving detection sensitivity. Therefore, the design and synthesis of fluorescent microsphere / degradation enzyme nanomaterials is key to this efficient immunochromatographic method. Currently, the design and synthesis of fluorescent microspheres / degradation enzymes-anti-thiabendazole monoclonal antibodies as signal probes and the simultaneous construction of dual-signal amplification immunochromatographic strips for thiabendazole detection have not been reported. Summary of the Invention

[0005] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art and to provide an immunochromatographic test strip based on fluorescent microspheres / degrading enzyme dual signals and a preparation method and application thereof.

[0006] The technical solutions of the present invention are as follows: The first aspect of the present invention provides an immunochromatographic test strip based on a fluorescent microsphere / degrading enzyme dual signal, wherein the immunochromatographic test strip uses a fluorescent microsphere / degrading enzyme nanocomposite material as a signal label; The preparation method of the fluorescent microsphere / decomposition enzyme nanocomposite material comprises the following steps: S1. Mix the fluorescent microspheres and ethanesulfonic acid solution evenly, then add EDC and NHS and mix; continue to add streptavidin solution and mix, centrifuge, and add PBS to obtain a streptavidin-fluorescent microsphere solution; dissolving a degrading enzyme in a buffer solution to obtain a degrading enzyme solution; dissolving biotin in a solvent, adding EDC and NHS to obtain a biotin solution; adding the biotin solution to the degrading enzyme solution and mixing, and removing unbound biotin after the reaction is completed to obtain a biotinylated degrading enzyme; S2. Mixing the biotinylated decomposition enzyme with the streptavidin-fluorescent microsphere solution, reacting the mixture, collecting the precipitate by centrifugation, washing the mixture, and drying the mixture to obtain a fluorescent microsphere / decomposition enzyme nanocomposite material.

[0007] Preferably, in step S1, the fluorescent microspheres are one or more of polystyrene fluorescent microspheres, magnetic fluorescent microspheres, fluorescent dye-labeled fluorescent microspheres, and biocompatible fluorescent microspheres; and the decomposition enzyme is one or more of catalase, peroxidase, and superoxide dismutase.

[0008] Preferably, in step S1, the volume ratio of the fluorescent microspheres, ethanesulfonic acid solution, EDC, NHS and streptavidin solution is 10-15 μL:700-800 μL:5 μL:5 μL:3-5 mL, and the added mass ratio of the degrading enzyme and biotin is 10-20:1-5.

[0009] In step S2, the ratio of adding the biotinylated degrading enzyme to adding the streptavidin-fluorescent microsphere solution is 600-800 μL: 200-300 μL.

[0010] Preferably, step S1 includes the following specific steps: The fluorescent microspheres were mixed with a pH 6-7, 0.045-0.055 M ethanesulfonic acid solution, and then vortexed, mixed, and ultrasonically stirred for 1-3 minutes to uniformly disperse the microspheres. Subsequently, 1 mg / mL EDC and 0.8-1.2 mg / mL NHS were added to the above solution, and the mixture was incubated in the dark at a constant temperature of 35-39°C for 25-35 minutes to obtain a mixed solution. Dilute streptavidin with 0.01 M PBS to 1.5-2.5 mg / mL to obtain a streptavidin solution; add the streptavidin solution to the mixed solution, vortex evenly, shake at 35-39°C for 1.5-2.5 hours, centrifuge at 10,000-14,000 r for 10-30 minutes, and add 0.01 M PBS to obtain a streptavidin-fluorescent microsphere solution; The degrading enzyme is dissolved in a buffer solution to obtain a degrading enzyme solution; biotin is dissolved in DMSO or water, and 0.8-1.2 mg / mL of EDC and 0.8-1.2 mg / mL of NHS are added to obtain a biotin solution; the biotin solution is added to the degrading enzyme solution, mixed, and placed on a shaker at room temperature for 6-10 hours. After the reaction is completed, unbound biotin is removed by dialysis to obtain a biotinylated degrading enzyme.

[0011] The immunochromatographic test strip uses fluorescent microspheres / lytic enzyme-labeled antibodies as signal probes. The preparation method of the fluorescent microspheres / lytic enzyme-labeled antibodies comprises: preparing the fluorescent microspheres / lytic enzyme nanocomposite material into a solution, mixing it with a solution of an antibody to be labeled, incubating, adding a blocking agent, and collecting a precipitate by centrifugation; and adding the precipitate to a resuspension to obtain a fluorescent microspheres / lytic enzyme-labeled antibody nanoprobe.

[0012] Preferably, the method for preparing the fluorescent microsphere / degrading enzyme labeled antibody comprises the following specific steps: The fluorescent microsphere / degrading enzyme nanocomposite material is prepared into a solution, and then 0.5-1.0 mL of the fluorescent microsphere / degrading enzyme nanocomposite material solution is mixed with 1-3 μL of a 0.5-2.0 mg / mL antibody solution to be labeled, and the mixture is shaken and incubated at room temperature for 20-60 min. A blocking agent is added and the mixture is further shaken at room temperature for 20-40 min. The mixture is centrifuged at 10,000-14,000 r for 10-30 min at 2-8° C., the supernatant is removed, and a resuspension solution is added to the precipitate for beating to obtain a fluorescent microsphere / degrading enzyme labeled antibody nanoprobe.

[0013] Preferably, in step S3, The blocking agent is one or more of bovine serum albumin, ovalbumin, polyvinyl alcohol, casein, and gelatin; The resuspension solution is one or more of phosphate buffer, Tris buffer, bovine serum albumin, Tween-20, Triton X-100, and sodium azide; The antibody to be labeled includes an anti-thiabendazole monoclonal antibody.

[0014] Preferably, the immunochromatographic test strip further comprises a sample pad and a nitrocellulose membrane, the nitrocellulose membrane is provided with a detection line and a quality inspection line, and the distance between the detection line and the quality inspection line is controlled to be 3 to 8 mm; The preparation method of the test line comprises: streaking 0.1-0.5 mg / mL of an antigen-carrier protein conjugate on a nitrocellulose membrane at a speed of 0.5-1.0 μL / cm to form a test line; the carrier protein is one or more of bovine serum albumin and ovalbumin; The preparation method of the quality inspection line comprises: streaking 0.5-1.0 mg / mL rabbit anti-pig IgG antibody on a nitrocellulose membrane at a speed of 0.6-1.2 μL / cm to form a quality inspection line.

[0015] The second aspect of the present invention provides a use of the immunochromatographic test strip in pesticide detection.

[0016] Preferably, the pesticide includes thiabendazole, and the method for detecting thiabendazole using the immunochromatographic test strip includes: The sample to be tested is mixed with a signal probe made of a fluorescent microsphere / degrading enzyme nanocomposite material for 5 to 10 minutes to obtain a mixed solution; the mixed solution is then dropped onto the sample pad of the immunochromatographic test strip and placed at 25 to 40° C. for 5 to 15 minutes; Use a fluorescent immunoassay reader to read the T and C values ​​for qualitative and semi-quantitative detection; when the T line has no peak and the C line has a peak, the result is positive; when both the T and C lines have peaks, the result is negative; after the reading is completed, carefully cut the T line, place it in a portable oxygen sensor and evacuate it, add H2O2 solution in a quantitative manner, wait 5 to 10 minutes, and use the instrument to read the oxygen content generated for quantitative detection.

[0017] The present invention has at least one of the following beneficial effects: In the immunochromatographic test strips involved in the present invention, fluorescent microspheres / degrading enzymes are used as signal labels, and fluorescent microspheres / degrading enzymes are combined with antibodies to the substance to be detected to form fluorescent microspheres / degrading enzyme-labeled antibodies as signal probes. The antibodies can specifically be anti-thiabendazole monoclonal antibodies, which can be used to detect thiabendazole in food. Compared with other immunochromatographic test strips based on labeling carriers such as colloidal gold for detecting thiabendazole in food, the new immunochromatographic test strips prepared by the present invention have the advantages of signal amplification, wide detection range, high sensitivity, fast response speed, good repeatability, and high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is an electron micrograph of the polystyrene fluorescent microsphere / catalase nanocomposite material prepared in Example 1.

[0019] Figure 2 This is the particle size distribution diagram of the polystyrene fluorescent microsphere / catalase nanocomposite material prepared in Example 1.

[0020] Figure 3 The fluorescence intensity of the polystyrene fluorescent microspheres / catalase nanocomposite prepared in Example 1.

[0021] Figure 4 This is a photo of the test strip prepared in Example 1 for detecting thiabendazole in the sample.

[0022] Figure 5 This is the sensitivity evaluation of the test strip prepared in Example 1.

[0023] Figure 6 This is the specificity evaluation of the test strip prepared in Example 1.

[0024] Figure 7 This is an electron micrograph of the biocompatible fluorescent microsphere / superoxide dismutase nanocomposite material prepared in Example 2.

[0025] Figure 8 This is the particle size distribution diagram of the biocompatible fluorescent microsphere / superoxide dismutase nanocomposite material prepared in Example 2.

[0026] Figure 9This is the fluorescence intensity of the biocompatible fluorescent microsphere / superoxide dismutase nanocomposite prepared in Example 2.

[0027] Figure 10 This is a photo of the test strip prepared in Example 2 for detecting thiabendazole in the sample.

[0028] Figure 11 This is the sensitivity evaluation of the test strip prepared in Example 2.

[0029] Figure 12 This is the specificity evaluation of the test strip prepared in Example 2. DETAILED DESCRIPTION

[0030] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0031] Example 1 A method for preparing an immunochromatographic test strip based on a dual signal of fluorescent microspheres / degrading enzymes, comprising the following steps: (1) The carboxyl groups of polystyrene fluorescent microspheres can be linked to the amino groups in the antibody. Therefore, the polystyrene fluorescent microspheres are first carboxylated. The specific method is as follows: Mix 15 μL of polystyrene fluorescent microspheres with 700 μL of ethanesulfonic acid (MES) solution (pH 6.5, 0.05 M). Vortex, mix, and ultrasonically stir for 2 minutes to evenly disperse the microspheres. Subsequently, add EDC (1 mg / mL, 5 μL) and NHS (1 mg / mL, 5 μL) to the above solution and incubate in the dark at a constant temperature (37°C) for 30 minutes.

[0032] Dilute streptavidin to 2 mg / mL with 0.01 M PBS, take 5 mL and add the above solution, vortex evenly, shake at 37°C for 2 hours, centrifuge at 12000 r for 20 minutes, and add 600 μL of 0.01 M PBS to complete the binding of streptavidin and polystyrene fluorescent microspheres.

[0033] Dissolve 0.1 mg of catalase in an appropriate buffer solution (0.01 M PBS, 0.5 mL) to obtain a catalase solution. Dissolve 20 μg of biotin in 0.3 mL of DMSO and activate biotin by adding EDC (1 mg / mL, 5 μL) and NHS (1 mg / mL, 5 μL), respectively. Add the activated biotin to the catalase solution and mix gently. Incubate the reaction on a shaker at room temperature for 8 hours. Remove unbound biotin by dialysis to form biotinylated catalase.

[0034] 800 μL of biotinylated catalase was mixed with 300 μL of streptavidin-polystyrene fluorescent microspheres and reacted at a constant temperature for 2 h. Finally, the precipitate was separated from the solution by centrifuge, washed three times with deionized water, and vacuum dried at 50 °C for 24 h to obtain the polystyrene fluorescent microsphere / catalase nanocomposite material.

[0035] (2) 1.0 mL of polystyrene fluorescent microspheres / catalase nanocomposite solution was mixed with 2 μL of anti-thiabendazole monoclonal antibody (1 mg / mL) solution, incubated with shaking at room temperature for 30 min, 100 μL of bovine serum albumin was added and continued to shake at room temperature for 30 min, centrifuged at 12000 r at 4 °C for 20 min, the supernatant was removed, and phosphate buffer was added to the precipitate for beating to obtain polystyrene fluorescent microspheres / catalase-anti-thiabendazole monoclonal antibody nanoprobes.

[0036] (3) Assembling immunochromatographic test strips: After pretreatment of the sample pad, a 0.3 mg / mL thiabendazole antigen-bovine serum albumin conjugate was streaked onto a nitrocellulose membrane at a speed of 0.8 μL / cm to form the detection zone. The thiabendazole antigen-bovine serum albumin conjugate was prepared by dissolving 1 mg of thiabendazole hapten in 0.5 mL of DMF, adding 20 μL of EDC (10 mg / mL) and 20 μL of NHS (10 mg / mL). The mixture was stirred at room temperature in the dark for 2 hours. The solution was then added dropwise to 5 mL of PBS (pH 8.5) containing 20 mg of bovine serum albumin. The reaction was allowed to react at room temperature for 4 hours. The reaction solution was dialyzed at 4°C for 3 days to remove free small molecules. A 0.8 mg / mL rabbit anti-swine IgG antibody was streaked onto the nitrocellulose membrane at a speed of 0.8 μL / cm to form the quality control zone. The distance between the two lines was controlled to be 3 mm. The streaked nitrocellulose membrane was then treated and assembled into strips to obtain the assembled immunochromatographic test strips.

[0037] (4) Detection method of thiabendazole in the sample to be tested: The method for detecting thiabendazole in a sample using the assembled immunochromatographic test strip includes: The sample to be tested was mixed with the probe prepared in step (2) in a centrifuge tube and incubated for 10 minutes. The sample was then dropped onto the sample pad and incubated at 37°C for 10 minutes. The T and C values ​​were read using a fluorescent immunoassay reader to achieve qualitative and semi-quantitative detection. When the T line had no peak and the C line had a peak, the result was positive; when both the T and C lines had peaks, the result was negative.

[0038] (5) Sensitivity and specificity of detection: Sensitivity: Mix the thiabendazole standard with the probe prepared in step (2) in a centrifuge tube and incubate for 8 minutes. Then, drop it onto the sample pad and incubate at 37°C for 10 minutes. The concentrations of the thiabendazole standard are 0, 10, 50, 100, 200, and 500 ng / mL, respectively. A standard curve is drawn based on the decrease in T-line fluorescence intensity to determine the detection limit and detection range.

[0039] Specificity: After the reading is completed, carefully cut the T-line, place it in a portable oxygen sensor, and evacuate it. A quantitative amount of H2O2 solution is then added. After waiting for 10 minutes, the generated oxygen content is read by the instrument for quantitative detection. Glyphosate, simazine, carbendazim, atrazine, and isoproturon were then selected as interfering substances at concentrations of 1000 ng / mL, and thiabendazole was set at 100 ng / mL to investigate its specificity. A mixed solution of these six substances was used as a mixed sample, and a blank was also used.

[0040] Figure 1 This is an electron microscope image of the polystyrene fluorescent microsphere / catalase nanocomposite prepared in this example. Figure 1 It can be shown that the material is spherical with relatively uniform size, which is consistent with the expected result.

[0041] Figure 2 The particle size distribution diagram of the polystyrene fluorescent microspheres / catalase nanocomposite prepared in this example is shown in FIG. Figure 2 It can be shown that the particle size of the material is basically concentrated in the range of 100-200 nm, with an average particle size of 151.15 nm.

[0042] Figure 3 The fluorescence intensity of the polystyrene fluorescent microspheres / catalase nanocomposite prepared in this example is given by Figure 3 This shows that the material has a large fluorescence signal at the emission wavelength of 600~640 nm, and the maximum fluorescence intensity of 9673.45 appears at 621 nm.

[0043] Figure 4 This is a physical picture of using immunochromatographic test strips to detect thiabendazole in the sample to be tested. Figure 4 It can be seen that the C lines of four of the test strips are fluorescent, indicating the effectiveness of these test strips; the first test strip does not contain thiabendazole, and the T line fluorescence is the strongest. Then, as the concentration of the added thiabendazole standard increases, the fluorescence value gradually decreases until it disappears, which is consistent with the expected result.

[0044] Figure 5 The sensitivity standard curve is 12.9 ng / mL, the detection limit is 34.5-171.9 ng / mL, and R 2 =0.99979, indicating that the standard curve fits well.

[0045] Figure 6 The results are specificity results, which show that the polystyrene fluorescent microspheres / catalase dual-signal detection thiabendazole immunochromatographic test strip of this embodiment has good specificity.

[0046] Example 2 A method for preparing an immunochromatographic test strip based on a dual signal of fluorescent microspheres / degrading enzymes, comprising the following steps: (1) The carboxyl group of the biocompatible fluorescent microspheres can be connected to the amino group in the antibody, so the biocompatible fluorescent microspheres are first carboxylated. The specific method is as follows: 10 μL of biocompatible fluorescent microspheres was mixed with 800 μL of ethanesulfonic acid (MES) solution (pH 6.5, 0.02 M). The mixture was then vortexed, mixed, and ultrasonically stirred for 3 minutes to uniformly disperse the microspheres. Subsequently, EDC (2 mg / mL, 5 μL) and NHS (2 mg / mL, 5 μL) were added to the above solution and incubated in the dark at a constant temperature (37°C) for 30 minutes.

[0047] Dilute streptavidin to 5 mg / mL with 0.01 M PBS, take 3 mL and add the above solution, vortex evenly, shake at 37°C for 2 hours, centrifuge at 14000 r for 10 minutes, and add 500 μL of 0.01 M PBS to complete the binding of streptavidin and biocompatible fluorescent microspheres.

[0048] Dissolve 0.3 mg of superoxide dismutase in an appropriate buffer solution (0.01 M PBS, 0.5 mL), dissolve 30 μg of biotin in 0.1 mL of DMSO, and activate biotin by adding EDC (2 mg / mL, 5 μL) and NHS (2 mg / mL, 5 μL), respectively. Add the activated biotin to the superoxide dismutase solution and mix gently. Place the reaction on a shaker at room temperature for 10 h. Remove unbound biotin by dialysis to form biotinylated superoxide dismutase.

[0049] 600 μL of biotinylated superoxide dismutase was mixed with 300 μL of streptavidin-biocompatible fluorescent microspheres and reacted at a constant temperature for 2 h. Finally, the precipitate was separated from the solution by centrifuge, washed twice with deionized water, and vacuum dried at 60 °C for 24 h to obtain the biocompatible fluorescent microsphere / superoxide dismutase nanocomposite material.

[0050] (2) 1.0 mL of biocompatible fluorescent microsphere / superoxide dismutase nanocomposite solution was mixed with 2 μL of anti-thiabendazole monoclonal antibody (1 mg / mL) solution, incubated with shaking at room temperature for 30 min, added with ovalbumin and continued to shake at room temperature for 30 min, centrifuged at 14000 r for 10 min at 4 °C, removed the supernatant, and added Tris buffer to the precipitate for beating to obtain biocompatible fluorescent microsphere / superoxide dismutase-anti-thiabendazole monoclonal antibody nanoprobe.

[0051] (3) Assembling immunochromatographic test strips: The sample pad was pretreated, and then 0.2 mg / mL of thiabendazole antigen-ovalbumin conjugate was streaked onto a nitrocellulose membrane at a speed of 0.5 μL / cm to form a detection zone. The preparation method of the thiabendazole antigen-bovine serum albumin conjugate included the following steps: 1 mg of thiabendazole hapten was dissolved in 0.5 mL of DMF, followed by the addition of EDC (10 mg / mL, 20 μL) and NHS (10 mg / mL, 20 μL). The mixture was stirred at room temperature in the dark for 2 hours, and then the solution was added dropwise to 5 mL of PBS solution (pH 8.5) containing 20 mg of BSA. The reaction was allowed to react at room temperature for 4 hours, and the reaction solution was dialyzed at 4°C for 3 days to remove free small molecules. 0.5 mg / mL of rabbit anti-swine IgG antibody was streaked onto the nitrocellulose membrane at a speed of 0.5 μL / cm to form a quality control zone. The distance between the two lines was controlled to be 3 mm. The streaked digested cellulose membrane was treated and assembled into strips to obtain assembled immunochromatographic test strips.

[0052] (4) Detection method of thiabendazole in the sample to be tested: The method for detecting thiabendazole in a sample using the assembled immunochromatographic test strip includes: The sample to be tested was mixed with the probe prepared in step (2) in a centrifuge tube and incubated for 10 minutes. The sample was then dropped onto the sample pad and incubated at 37°C for 10 minutes. The T and C values ​​were read using a fluorescent immunoassay reader to achieve qualitative and semi-quantitative detection. When the T line had no peak and the C line had a peak, the result was positive; when both the T and C lines had peaks, the result was negative.

[0053] (5) Sensitivity and specificity of detection: Sensitivity: Mix the thiabendazole standard with the probe prepared in step (2) in a centrifuge tube and incubate for 8 minutes. Then, drop it onto the sample pad and incubate at 37°C for 10 minutes. The concentrations of the thiabendazole standard are 0, 10, 50, 100, 200, and 500 ng / mL, respectively. A standard curve is drawn based on the decrease in T-line fluorescence intensity to determine the detection limit and detection range.

[0054] Specificity: After the reading is completed, carefully cut the T-line, place it in a portable oxygen sensor, and evacuate it. A quantitative amount of H2O2 solution is then added. After waiting for 10 minutes, the generated oxygen content is read by the instrument for quantitative detection. Glyphosate, simazine, carbendazim, atrazine, and isoproturon were then selected as interfering substances at concentrations of 1000 ng / mL, and thiabendazole was set at 100 ng / mL to investigate its specificity. A mixed solution of these six substances was used as a mixed sample, and a blank was also used.

[0055] Figure 7 This is an electron microscope image of the polystyrene fluorescent microsphere / catalase nanocomposite prepared in this example. Figure 7 It can be shown that the material is spherical with relatively uniform size, which is consistent with the expected result.

[0056] Figure 8 The particle size distribution diagram of the polystyrene fluorescent microspheres / catalase nanocomposite prepared in this example is shown in FIG. Figure 8 It can be shown that the particle size of the material is concentrated in the range of 100-200 nm, with an average particle size of 147.09 nm.

[0057] Figure 9 The fluorescence intensity of the polystyrene fluorescent microspheres / catalase nanocomposite prepared in this example is given by Figure 9 This indicates that the material has a large fluorescence signal at the emission wavelength of 600~640 nm, and the maximum fluorescence intensity of 8873.16 occurs at 620 nm.

[0058] Figure 10 This is a physical picture of using immunochromatographic test strips to detect thiabendazole in the sample to be tested. Figure 10 It can be seen that the C lines of four of the test strips are fluorescent, indicating the effectiveness of these test strips; the first test strip does not contain thiabendazole, and the T line fluorescence is the strongest. Then, as the concentration of the added thiabendazole standard increases, the fluorescence value gradually decreases until it disappears, which is consistent with the expected result.

[0059] Figure 11 The sensitivity standard curve is 18.3 ng / mL, the detection range is 36-178.2 ng / mL, and R 2=0.99865, indicating that the standard curve fits well.

[0060] Figure 12 The results are specificity results, which show that the polystyrene fluorescent microspheres / catalase dual-signal detection thiabendazole immunochromatographic test strip of this embodiment has good specificity.

[0061] In summary, the immunochromatographic test strips prepared in Examples 1 and 2 of the present invention have the characteristics of high accuracy, wide detection range, and low detection limit for the detection of thiabendazole. At the same time, they exhibit good recovery rate and high stability, indicating that the prepared immunochromatographic test strips have excellent practical application value.

[0062] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. An immunochromatographic test strip based on fluorescent microspheres / degrading enzyme dual signals, characterized in that: The immunochromatographic test strip uses fluorescent microsphere / degrading enzyme nanocomposite material as a signal label; The preparation method of the fluorescent microsphere / decomposition enzyme nanocomposite material comprises the following steps: S1. Mix the fluorescent microspheres and ethanesulfonic acid solution evenly, then add EDC and NHS and mix; continue to add streptavidin solution and mix, centrifuge, and add PBS to obtain a streptavidin-fluorescent microsphere solution; dissolving a degrading enzyme in a buffer solution to obtain a degrading enzyme solution; dissolving biotin in a solvent, adding EDC and NHS to obtain a biotin solution; adding the biotin solution to the degrading enzyme solution and mixing, and removing unbound biotin after the reaction is completed to obtain a biotinylated degrading enzyme; S2. Mixing the biotinylated decomposition enzyme with the streptavidin-fluorescent microsphere solution, reacting the mixture, collecting the precipitate by centrifugation, washing the mixture, and drying the mixture to obtain a fluorescent microsphere / decomposition enzyme nanocomposite material.

2. The immunochromatographic test strip according to claim 1, wherein In step S1, the fluorescent microspheres are one or more of polystyrene fluorescent microspheres, magnetic fluorescent microspheres, fluorescent dye-labeled fluorescent microspheres, and biocompatible fluorescent microspheres; and the decomposition enzymes are one or more of catalase, peroxidase, and superoxide dismutase.

3. The immunochromatographic test strip according to claim 1, wherein In step S1, the volume ratio of the fluorescent microspheres, ethanesulfonic acid solution, EDC, NHS, and streptavidin solution is 10-15 μL:700-800 μL:5 μL:5 μL:3-5 mL, and the mass ratio of the added degrading enzyme and biotin is 10-20:1-5; In step S2, the ratio of the biotinylated degrading enzyme to the streptavidin-fluorescent microsphere solution is 600-800:200-300.

4. The immunochromatographic test strip according to claim 1, wherein Step S1 includes the following specific steps: The fluorescent microspheres were mixed with a pH 6-7, 0.045-0.055 M ethanesulfonic acid solution, and then vortexed, mixed, and ultrasonically stirred for 1-3 min to uniformly disperse the microspheres. Subsequently, EDC at a concentration of 1 mg / mL and NHS at a concentration of 0.8-1.2 mg / mL were added to the above solution, and the mixture was incubated in the dark at a constant temperature of 35-39°C for 25-35 min to obtain a mixed solution. Dilute streptavidin with 0.01 M PBS to 1.5-2.5 mg / mL to obtain a streptavidin solution; Add the streptavidin solution to the mixed solution, vortex evenly, shake at 35-39°C for 1.5-2.5 hours, centrifuge at 10,000-14,000 r for 10-30 minutes, and add 0.01 M PBS to obtain a streptavidin-fluorescent microsphere solution; The degrading enzyme is dissolved in a buffer solution to obtain a degrading enzyme solution; biotin is dissolved in DMSO or water, and 0.8-1.2 mg / mL of EDC and 0.8-1.2 mg / mL of NHS are added to obtain a biotin solution; the biotin solution is added to the degrading enzyme solution, mixed, and placed on a shaker at room temperature for 6-10 hours. After the reaction is completed, unbound biotin is removed by dialysis to obtain a biotinylated degrading enzyme.

5. The immunochromatographic test strip according to claim 1, characterized in that The immunochromatographic test strip uses fluorescent microspheres / lytic enzyme-labeled antibodies as signal probes. The preparation method of the fluorescent microspheres / lytic enzyme-labeled antibodies comprises: preparing the fluorescent microspheres / lytic enzyme nanocomposite material into a solution, mixing it with a solution of an antibody to be labeled, incubating, adding a blocking agent, and collecting a precipitate by centrifugation; and adding the precipitate to a resuspension to obtain a fluorescent microspheres / lytic enzyme-labeled antibody nanoprobe.

6. The immunochromatographic test strip according to claim 5, characterized in that The preparation method of the fluorescent microsphere / degradation enzyme labeled antibody comprises the following specific steps: The fluorescent microsphere / degrading enzyme nanocomposite material is prepared into a solution, and then 0.5-1.0 mL of the fluorescent microsphere / degrading enzyme nanocomposite material solution is mixed with 1-3 μL of a 0.5-2.0 mg / mL antibody solution to be labeled, and the mixture is shaken and incubated at room temperature for 20-60 min. A blocking agent is added and the mixture is further shaken at room temperature for 20-40 min. The mixture is centrifuged at 10,000-14,000 r at 2-8° C. for 10-30 min, the supernatant is removed, and a resuspension solution is added to the precipitate for beating to obtain a fluorescent microsphere / degrading enzyme labeled antibody nanoprobe.

7. The immunochromatographic test strip according to claim 5, characterized in that The blocking agent is one or more of bovine serum albumin, ovalbumin, polyvinyl alcohol, casein, and gelatin; The resuspension solution is one or more of phosphate buffer, Tris buffer, bovine serum albumin, Tween-20, Triton X-100, and sodium azide; The antibody to be labeled includes an anti-thiabendazole monoclonal antibody.

8. The immunochromatographic test strip according to claim 1, characterized in that The immunochromatographic test strip further comprises a sample pad and a nitrocellulose membrane, wherein a detection line and a quality inspection line are provided on the nitrocellulose membrane, and the distance between the detection line and the quality inspection line is controlled to be 3 to 8 mm; The preparation method of the test line comprises: streaking 0.1-0.5 mg / mL of an antigen-carrier protein conjugate on a nitrocellulose membrane at a speed of 0.5-1.0 μL / cm to form a test line; the carrier protein is one or more of bovine serum albumin and ovalbumin; The preparation method of the quality inspection line comprises: streaking 0.5-1.0 mg / mL rabbit anti-pig IgG antibody on a nitrocellulose membrane at a speed of 0.6-1.2 μL / cm to form a quality inspection line.

9. Use of the immunochromatographic test strip according to any one of claims 1 to 8 in pesticide detection.

10. The use according to claim 9, characterized in that The pesticide includes thiabendazole, and the method for detecting thiabendazole using the immunochromatographic test strip includes: The sample to be tested is mixed with a signal probe made of a fluorescent microsphere / degrading enzyme nanocomposite material for 5 to 10 minutes to obtain a mixed solution; the mixed solution is then dropped onto the sample pad of the immunochromatographic test strip and placed at 25 to 40° C. for 5 to 15 minutes; Use a fluorescent immunoassay reader to read the T and C values ​​for qualitative and semi-quantitative detection; when the T line has no peak and the C line has a peak, the result is positive; when both the T and C lines have peaks, the result is negative; after the reading is completed, carefully cut the T line, place it in a portable oxygen sensor and evacuate it, add H2O2 solution in a quantitative manner, wait 5 to 10 minutes, and use the instrument to read the oxygen content generated for quantitative detection.

Images