Immunochromatography time-resolved fluorescence kit for polystyrene micro-nanoplastics and its use

Abstract

This invention provides an immunochromatographic time-resolved fluorescence kit for polystyrene micro-nanoplastics and its use. [Solution] The solution comprises a fluorescent test paper strip and a sample reaction flask containing a lyophilized anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres. The fluorescent test paper strip includes a bottom plate, on which an absorbent pad, a detection pad, and a sample pad are attached from top to bottom on the adhesive surface of the bottom plate, with adjacent pads overlapping and connected at connection points. The detection pad uses a nitrocellulose membrane as its base pad, with a lateral quality control line and a detection line provided on the nitrocellulose membrane from top to bottom. The quality control line is coated with an immunoanti-mouse polyclonal antibody, and the detection line is coated with a polystyrene-hemocyanin conjugate.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological materials, and specifically relates to an immunochromatography time-resolved fluorescence kit for polystyrene micro-nanoplastics and its use.

Background Art

[0002] Microplastics are novel environmental pollutants that adsorb heavy metals and persistent organic pollutants and move and transform in the environment. Microplastics are taken into the animal body and are likely to release the toxic and harmful substances they carry, damaging the growth, reproduction, gene expression, etc. of organisms.

[0003] Currently, the main quantitative analysis methods for microplastics are mainly microscopic infrared spectroscopy, microscopic Raman spectroscopy, visual method, etc. The visual method uses an artificial counting method to count the number of microplastic particles, select and weigh all microplastics, which is not only time-consuming but also prone to errors in operation. Microscopic infrared spectroscopy and microscopic Raman spectroscopy are almost the same as the visual method in the quantitative method, but by using infrared spectra and Raman spectra instead of identifying particles with the naked eye, the accuracy of analysis is greatly improved. Infrared and Raman spectra must identify microplastics one by one, and the surface of the identified microplastics cannot be contaminated with organic pollutants, requiring high pretreatment technology and a large amount of analysis time.

[0004] Therefore, an immunochromatography time-resolved fluorescence kit for polystyrene micro-nanoplastics and its use have been urgently proposed.

Summary of the Invention

Problems to be Solved by the Invention

[0005] To address the shortcomings of the prior art, the present invention provides an immunochromatographic time-resolved fluorescence kit for polystyrene micro-nanoplastics and its use. [Means for solving the problem]

[0006] To solve the above technical problems, the present invention provides the following technical means.

[0007] The first object of the present invention is a sample reaction flask comprising a fluorescent test strip and a lyophilized anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres, wherein the fluorescent test strip includes a bottom plate, and an absorbent pad, a detection pad, and a sample pad are attached to the adhesive surface of the bottom plate in order from top to bottom, with adjacent pads overlapping and connected at connection points, the detection pad having a nitrocellulose membrane as a base pad, the nitrocellulose membrane having a lateral quality control line and a detection line from top to bottom, the quality control line being coated with an immunoanti-mouse polyclonal antibody, the detection line being coated with a polystyrene-hemocyanin conjugate, the anti-polystyrene monoclonal antibody being secreted from the hybridoma cell line PS-17, and the deposit number of the hybridoma cell line PS-17 is CCTCC The present invention provides an immunochromatographic time-resolved fluorescence kit for polystyrene micro-nanoplastics, characterized by its product number: C2024145, deposit date: May 10, 2024, depositary institution: China Typical Culture Depository Center, depositary institution address: Wuhan University Campus, 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0008] Preferably, the lyophilized product of the anti-polystyrene monoclonal antibody labeled with the quantum dot fluorescent microspheres is Step S11 involves mixing an anti-polystyrene monoclonal antibody with an activated quantum dot fluorescent microsphere labeling reagent in borate buffer, shaking, centrifugation, redissolution, and blocking to obtain an anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres, wherein 1 mL of activated quantum dot fluorescent microsphere labeling reagent is coupled to 40 pg to 90 ug of anti-polystyrene monoclonal antibody. Step S12 involves redissolving the anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres prepared in step S11 in 0.01 m³ / L, pH 7.4 phosphate buffer containing 1.5% (m / v) trehalose and 2% ( / v) bovine serum albumin, and lyophilizing the mixture in a lyophilizer to obtain a lyophilized product of the anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres.

[0009] Preferably, the method for activating the quantum dot fluorescent microsphere labeling reagent activated in step S11 includes the steps of: dispersing the quantum dot microsphere labeling reagent in pH 8.2, 0.2 M borate buffer by sonication; slowly adding the carbodiimide solution; activating by shaking at room temperature; removing the supernatant by centrifugation; redissolving in pH 8.2, 0.2 M borate buffer; and preparing for use, with the activation time set to 15-30 minutes.

[0010] Preferably, the sample reaction flask is a 1-5 mL bayonet flask, and the content of the lyophilized anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres in the sample reaction flask is 100-200 ng.

[0011] Preferably, the fluorescent test paper strip is Step S21 involves cutting absorbent paper to obtain an absorbent pad, In the manufacturing step S22 of the detection pad, a coating solution of polystyrene-hemocyanin conjugate is prepared with a coating buffer to a concentration of 0.2 to 0.5 mg / mL, and the coating solution is coated laterally onto the nitrocellulose membrane by line spraying at a position 15 to 20 m away from the upper edge of the nitrocellulose membrane to obtain a detection line. After that, it is dried at 37 to 40°C for 60 to 120 minutes, and the amount of polystyrene-hemocyanin conjugate coating required per 1 cm of detection line is set to 80 to 400 ng. Step S22 involves preparing a coating solution with a concentration of 0.2-0.5 mg / mL using an immunosuppressant mouse polyclonal antibody in a coating buffer, coating a nitrocellulose membrane laterally with the coating solution using a line spray method at a distance of 5-10 m from the detection line to obtain a quality control line, drying the membrane at 37-40°C for 60-120 minutes, and ensuring that the coating amount of immunosuppressant mouse polyclonal antibody per 1 cm of the quality control line is 100-300 ng. Step S23 of the sample pad manufacturing process involves wetting a glass fiber film in a blocking solution, removing it, drying it for 10-16 hours under conditions of 37-40°C to obtain a sample pad, and then storing it in a drying oven at room temperature. The fluorescent test paper strip is manufactured by the assembly step S24, in which an absorbent pad, a detection pad, and a sample pad are attached to one side of a cardboard sheet in order from top to bottom, and adjacent pads are connected by overlapping them at connection points, with an overlap length of 1 to 3 mm.

[0012] Preferably, the absorbent pad in the fluorescent test paper strip has a length of 16-18 mm and a width of 3-4 mm, the detection pad has a length of 18-30 mm and a width of 3-4 mm, the sample pad has a length of 10-12 mm and a width of 3-4 mm, the distance between the detection line on the detection pad in the fluorescent test paper strip and the upper edge of the nitrocellulose film is 15-20 mm, and the distance between the quality control line and the detection line is 5-10 mm.

[0013] Preferably, the coating buffer for the polystyrene-hemocyanin conjugate in step S22 is prepared by adding water to 1 g of bovine serum albumin, 0.02 g of sodium azide, 0.8 g of sodium chloride, 0.29 g of disodium hydrogen phosphate dodecyl, 0.02 g of potassium chloride, and 0.02 g of potassium dihydrogen phosphate to a final volume of 100 mL. The coating buffer for immunoanti-mouse polyclonal antibodies is prepared by adding water to 0.02 g of sodium azide, 0.8 g of sodium chloride, 0.29 g of disodium hydrogen dodecyl phosphate, 0.02 g of potassium chloride, and 0.02 g of potassium dihydrogen phosphate to a total volume of 100 ml.

[0014] Preferably, the blocking solution in step S23 is prepared by adding water to 2 g of ovalbumin, 2 g of sucrose, 0.02 g of sodium azide, 0.8 g of sodium chloride, 0.29 g of disodium hydrogen phosphate dodecyl, 0.02 g of potassium chloride, 0.02 g of potassium dihydrogen phosphate, and 0.5 g of Zeen-20 to a final volume of 100 mL.

[0015] Preferably, the kit further comprises a sample diluent and a sample diluent pipette, wherein the sample diluent is a Tzeen-20 aqueous solution with a volume fraction of 0.01 to 0.30%.

[0016] A second object of the present invention is to provide the use of an immunochromatographic time-resolved fluorescence kit for polystyrene micro-nanoplastics, which is used for detecting the content of polystyrene micro-nanoplastics. [Effects of the Invention]

[0017] Compared to the prior art, the present invention has the following beneficial effects.

[0018] The immunochromatography time-resolved fluorescence kit according to the present invention can achieve quantitative detection of PS, has strong specificity, high sensitivity, simple sample pretreatment, low environmental pollution, and is suitable for mass detection on-site. In particular, it has advantages such as removing the high background in general fluorescence measurement, has a great potential for development in non-radioactive immunoassay, and can be used in fields such as food, environment, and medicine.

[0019] Regarding cell deposit, The anti-polystyrene monoclonal antibody according to the present invention is secreted from the hybridoma cell line PS-17. The hybridoma cell line PS-17 was obtained by screening by the present inventor. The deposit number of the hybridoma cell line PS-17 is CCTCC NO:C2024145, the deposit date is May 10, 2024, the deposit institution is the China Center for Type Culture Collection, and the deposit institution address is within the campus of Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

Brief Description of the Drawings

[0020] [Figure 1] It is a schematic configuration diagram of the fluorescence test strip according to Example 1 of the present invention. [Figure 2] It is a schematic diagram of the SDS-PAGE electrophoresis of PS-BSA and PS-KLH according to Example 2 of the present invention.

Modes for Carrying Out the Invention

[0021] Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. It should be understood that the preferred embodiments described here are only for explaining and interpreting the present invention and do not limit the present invention.

[0022] (Example 1) Manufacture of an immunochromatography time-resolved fluorescence kit for polystyrene micro / nanoplastics The immunochromatography time-resolved fluorescence kit for polystyrene micro / nanoplastics according to this embodiment includes a fluorescence test strip (Figure 1) and a sample reaction flask containing a lyophilized product of an anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres. The fluorescence test strip includes a bottom plate, and a water-absorbing pad 1, a detection pad 2, and a sample pad 3 are sequentially pasted on the adhesive surface of the bottom plate from top to bottom. Each adjacent pad is connected by overlapping at the connection point. The detection pad 2 uses a nitrocellulose membrane as the base pad, and a horizontal quality control line 4 and a detection line 5 are provided on the nitrocellulose membrane from top to bottom. The quality control line 4 is coated with an immune anti-mouse polyclonal antibody, and the detection line 5 is coated with a polystyrene-hemocyanin conjugate. The anti-polystyrene monoclonal antibody is secreted from the hybridoma cell line PS-17. The deposit number of the hybridoma cell line PS-17 is CCTCC NO: C2024145, the deposit date is May 10, 2024, the depository is the China Center for Type Culture Collection, and the depository address is within the campus of Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0023] In this embodiment, the lyophilized product of the anti-polystyrene monoclonal antibody labeled with the quantum dot fluorescent microspheres is manufactured through the following steps S11 and S12.

[0024] In step S11, the anti-polystyrene monoclonal antibody and the activated quantum dot fluorescent microsphere labeling reagent are mixed in a borate buffer solution, shaken for reaction, and after centrifugation, redissolution, and blocking steps, an anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres is obtained, and 1 mL of the activated quantum dot fluorescent microsphere labeling reagent is coupled to 40 pg to 90 μg (in this embodiment, it may be 40 pg, 20 μg, or 90 μg) of the anti-polystyrene monoclonal antibody.

[0025] The method for activating the activated quantum dot fluorescent microsphere labeling reagent includes the steps of: dispersing the quantum dot microsphere labeling reagent sonically in pH 8.2, 0.2 M borate buffer; slowly adding the carbodiimide solution; activating by shaking at room temperature; removing the supernatant by centrifugation; redissolving in pH 8.2, 0.2 M borate buffer; and preparing for use, with the activation time set to 15-30 min (in this embodiment, it may be 15, 20, or 30 min). The sample reaction flask is a 1-5 mL (in this embodiment, it may be 1, 3, or 4 mL) bayonet flask, and the content of the lyophilized anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres in the sample reaction flask is 100-200 ng (in this embodiment, it may be 100, 150, or 200 ng).

[0026] In step S12, the anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres prepared in step S11 is redissolved in a 0.01 m³ / L, pH 7.4 phosphate buffer containing 1.5% (m / v) trehalose and 2% ( / v) bovine serum albumin. The mixture is then freeze-dried in a lyophilizer to obtain a lyophilized product of the anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres.

[0027] In this embodiment, the fluorescent test paper strip is manufactured in the following steps S21 to S24.

[0028] In step S21, absorbent paper is cut to obtain an absorbent pad.

[0029] In the manufacturing step S22 of the detection pad, a coating solution is prepared using a coating buffer to a concentration of 0.2 to 0.5 mg / mL (in this example, it may be 0.2, 0.3, or 0.5 mg / m) using polystyrene-hemocyanin conjugate. The coating solution is then coated laterally onto the nitrocellulose membrane by line spraying at a distance of 15 to 20 m (in this example, it may be 15, 18, or 20 m) from the upper edge of the nitrocellulose membrane to obtain a detection line. After that, the membrane is dried at 37 to 40°C for 60 to 120 minutes (in this example, it may be dried at 37°C for 60 minutes and then at 38°C for 80 minutes, or at 40°C for 120 minutes). The required amount of polystyrene-hemocyanin conjugate coating per 1 cm of detection line is 80 to 400 ng (in this example, it may be 80, 200, or 400 ng), and the coating buffer for the polystyrene-hemocyanin conjugate is prepared by adding water to 100 mL of 1 g of bovine serum albumin, 0.02 g of sodium azide, 0.8 g of sodium chloride, 0.29 g of disodium hydrogen dodecyl phosphate, 0.02 g of potassium chloride, and 0.02 g of potassium dihydrogen phosphate, and then adjusting the volume to 100 mL. An immunoanti-mouse polyclonal antibody is prepared in a coating buffer to a concentration of 0.2 to 0.5 mg / mL (in this example, it may be 0.2, 0.3, or 0.5 mg / mL) and the coating solution is applied laterally to a nitrocellulose membrane by line spraying at a distance of 5 to 10 m (in this example, it may be 5, 8, or 10 m) from the detection line to obtain a quality control line. After that, the membrane is dried at 37 to 40°C for 60 to 120 minutes (in this example, it may be dried at 37°C for 60 minutes and then at 38°C for 80 minutes, or at 40°C for 120 minutes). The amount of immunosuppressive mouse polyclonal antibody coating per 1 cm of quality control line was set to 100-300 ng (in this example, it may be 100, 200, or 300 ng), and the coating buffer for immunosuppressive mouse polyclonal antibody was prepared by adding water to 0.02 g of sodium azide, 0.8 g of sodium chloride, 0.29 g of disodium hydrogen dodecyl phosphate, 0.02 g of potassium chloride, and 0.02 g of potassium dihydrogen phosphate to a final volume of 100 ml. In the sample pad manufacturing step S23, the glass fiber membrane is wetted in the blocking solution, removed, and dried for 10 to 16 hours under conditions of 37 to 40°C (in this example, it may be dried for 10 hours under conditions of 37°C and then for 14 hours under conditions of 38°C, or for 16 hours under conditions of 40°C) to obtain the sample pad. After obtaining the sample pad, it is stored at room temperature in a drying oven. The blocking solution is prepared by adding water to 2 g of ovalbumin, 2 g of sucrose, 0.02 g of sodium azide, 0.8 g of sodium chloride, 0.29 g of disodium hydrogen phosphate dodecyl, 0.02 g of potassium chloride, 0.02 g of potassium dihydrogen phosphate, and 0.5 g of Zeen-20 to a final volume of 100 mL. In the assembly step S24 of the fluorescent test strip, an absorbent pad, a detection pad, and a sample pad are attached to one side of the cardboard in order from top to bottom, and adjacent pads are connected by overlapping them at the connection points, with an overlap length of 1 to 3 mm (in this embodiment, it may be 1, 2, or 3 mm) to obtain a fluorescent test strip.

[0030] In this embodiment, the absorbent pad in the fluorescent test paper strip has a length of 16 to 18 mm (in this embodiment, it may be 16, 17, or 18 mm) and a width of 3 to 4 mm (in this embodiment, it may be 3, 3.5, or 4 mm), the detection pad has a length of 18 to 30 mm (in this embodiment, it may be 18, 25, or 30 mm) and a width of 3 to 4 mm (in this embodiment, it may be 3, 3.5, or 4 mm), the sample pad has a length of 10 to 12 mm (in this embodiment, it may be 10, 11, or 12 mm) and a width of 3 to 4 mm (in this embodiment, it may be 3, 3.5, or 4 mm), the distance between the detection line on the detection pad in the fluorescent test paper strip and the upper edge of the nitrocellulose film is 15 to 20 mm (in this embodiment, it may be 15, 18, or 20 mm), and the distance between the quality control line and the detection line is 5 to 10 mm (in this embodiment, it may be 5, 6, or 10 mm).

[0031] In this example, the kit further comprises a sample diluent, which is a Tzeen-20 aqueous solution with a volume fraction of 0.01 to 0.30%.

[0032] (Example 2) Manufacturing of anti-polystyrene monoclonal antibodies (1) For the coupling of microplastic (PS-CHOOH) and coupling protein (BSA / KLH), the microplastic, coupling buffer solution (Polylink coupling buffer), coupling carbodiimide (Polylink EDAC), and coupling buffer solution were cooled to room temperature. 12.5 mg of microplastic was placed in a 1.5 mL centrifuge tube and centrifuged at 500-1000 rpm for 5-10 mins. The supernatant was discarded, 0.4 mL of coupling buffer solution was added and the mixture was resuspended. It was centrifuged again, the supernatant was discarded, and another 0.17 mL of coupling buffer solution was added and the mixture was resuspended. 10 mg of polylink EDAC was dissolved in 50 μL of coupling buffer solution to prepare a 200 mg / mL EDAC solution, which was to be prepared as needed. 20 μL of the EDAC solution was added to the 0.17 mL coupling buffer solution suspension, and the mixture was fixed to a rotating apparatus and mixed uniformly at room temperature for 15 mins for activation. 200-500 μg of coupling protein (BSA / KLH) was added and the mixture was fixed to a rotating apparatus and mixed uniformly at room temperature for 30-60 min. (The protein was dissolved in coupling buffer solution at a concentration of 1-5 mg / ml.) The mixture was centrifuged at 500-1000 rpm for 10 minutes, and the supernatant was aspirated to detect the amount of coupling protein (BSA / KLH). 0.4 mL of polylink wash / storage buffer was added to resuspend the mixture, and the precipitate at the bottom of the tube was either PS-BSA or PS-KLH. Finally, the mixture was stored at 4°C. SDS-PAGE analysis (4% concentrated gel and 7.5% separated gel) was performed. The detection results of reduced SDS-PAGE are shown in Figure 2, demonstrating that the coupling of PS-KLH and PS-BSA was successful.

[0033] (2) For the production of monoclonal antibodies, female Balb / c mice aged 6-8 weeks were selected and immunohistochemically injected with the produced PS-BSA artificial antigen. Adult Balb / c mice received primary immunization by subcutaneous administration (in the area between the shoulders), followed by booster immunization every two weeks. Blood samples were collected at week 0 (before immunization), week 8 (two weeks after the fourth reinforcement injection), week 10 (before the third reinforcement injection), week 12, and week 14. Serum separation was performed to obtain B lymphocytes, RNA was extracted, and transcribed into a cDNA library. By amplifying the cDNA again, specific gene fragments were obtained, and these gene fragments were recombined into phages to construct a nano-antibody gene library. Next, specific antibody screening was performed to obtain antibodies that met the requirements. The PS-BSA artificial antigen was immobilized on a carrier and then interacted with the phage library. Subsequently, the surface of the carrier was washed to wash away phages that were not bound or were nonspecifically bound, and the binding was further broken with a strong acid to obtain a positive phage solution. After repeating the process two to three times, a monoclonal antibody that meets the requirements can be obtained.

[0034] The scheme for immunizing mice by injecting the PS-BSA artificial antigen is shown in Table 1. We observed the specific responses to the immune system of Balb / c mice and investigated the activation mechanism of antibody-specific immune responses.

[0035] Table 1 Scheme of immunized mice [Table 1]

[0036] The obtained monoclonal antibody includes a heavy chain variable region and a light chain variable region, both of which are composed of a determination cluster complementary region and a framework region, and both of which are composed of CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 in the heavy chain variable region is shown at positions 50-54 of SEQ ID No. 1. The amino acid sequence of CDR2 in the heavy chain variable region is shown at positions 69-85 of SEQ ID No. 1. The amino acid sequence of CDR3 in the heavy chain variable region is shown at positions 118-128 of SEQ ID No. 1. The amino acid sequence of CDR1 in the light chain variable region is shown at positions 44-54 of SEQ ID No. 2. The amino acid sequence of the light chain variable region CDR2 is shown from position 70 to 76 of SEQ ID No. 2. The amino acid sequence of CDR3 in the light chain variable region is shown at positions 109-117 of SEQ ID No. 2.

[0037] The amino acid sequence of the antibody's heavy chain variable region is shown in SEQ ID No. 1 below.

[0038] MEWIWIFLFILSGTAGVHSQVQLQQSGAELARPGASVKLSCKASGYTFTDYYINWVKQRTGQGLEWIGEIYPGSGNTYYNEKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCARSEIYGIYYFDYWGQGTTLTVSS.

[0039] The amino acid sequence of the antibody's light chain variable region is shown in SEQ ID No. 2 below.

[0040] METHSQVFVYMLLWLSGVEGDIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPYTFGGGTKLEIK.

[0041] (Example 3) Use of immunochromatographic time-resolved fluorescence kits for polystyrene micro-nanoplastics.

[0042] The specific principle is as follows: After pre-treating the sample to be detected to obtain a sample solution, it is placed in a sample reaction flask, mixed uniformly, a fluorescent test strip is inserted, and the mixture is reacted at 37°C for 6 to 10 minutes. The solution is then detected using a time-resolved fluorescence tester, and the ratio of the fluorescence intensity of the detection line (T) to the fluorescence intensity of the quality control line (C) is obtained using the fluorescence immunochromatography test strip. Based on the relationship curve between the ratio of the fluorescence intensity of the detection line to the fluorescence intensity of the quality control line and the polystyrene concentration, the polystyrene content in the sample solution is obtained, and finally, the polystyrene content in the sample is calculated.

[0043] Water and soil samples were collected from oceans, rivers, and lakes. Wastewater samples from textile companies were collected. Food samples were collected from pillows or tables. After pretreatment, the collected samples were quantitatively detected using test strips.

[0044] The following steps were used for preprocessing.

[0045] (1) For liquid samples, such as water samples, each sample was centrifuged at 17,000 rpm for 3 minutes, the supernatant was digested and extracted, and the digested extract was diluted with the sample diluent and used for immunochromatographic quantitative detection using a test strip.

[0046] (2) For non-liquid samples, it was necessary to digest the sample, extract microplastics, dilute it with a diluent, and then perform immunochromatographic quantitative detection.

[0047] As an example, soil samples (detection by adding a standard substance) were used. Deionized water was added to the soil sample (1:1 w / v), and then vortexed for 10 seconds. The slurry mixture was centrifuged at 17,000 rpm for 3 minutes, and the supernatant was used as a specific matrix extract for standard substance recovery analysis. Briefly, 50 μL of polystyrene (25 μg) was added to 950 μL of matrix or matrix extract, mixed, and incubated at 37°C for 16 hours. After incubation, the sample was centrifuged at 5,500 rpm for 3 minutes, and the supernatant was discarded. The polystyrene particles were suspended in PBST washing buffer and washed four times (incubated for 3 minutes after each wash). In all measurements, the same matrix sample was used. The washing solution PBST was used as the control matrix. Subsequently, the washed polystyrene particles were subjected to immunochromatographic measurements in the same manner as above.

[0048] According to the above scheme, the relationship curve between the ratio (T / C) of the fluorescence intensity of the detection line (T) to the fluorescence intensity of the quality control line (C) measured by the fluorescent test paper strip and the polystyrene concentration was obtained by the following method.

[0049] (1) Prepare a series of polystyrene standard solutions of different concentrations, (2) Appropriate amounts of polystyrene standard solutions of each concentration were added to the sample reaction flasks, mixed uniformly, and fluorescent test strips were inserted. The mixture was reacted at 37°C for 6 to 10 minutes. The mixture was then detected using a time-resolved fluorescence immunoassay analyzer to obtain the detection line (T) and the time-resolved fluorescence intensity (C) of the quality control line using each immunochromatographic time-resolved fluorescence test strip. The ratio (T / C) of the detection line fluorescence intensity to the quality control line fluorescence intensity using each immunochromatographic time-resolved fluorescence test strip was obtained, and a relationship curve between the ratio of the detection line fluorescence intensity to the quality control line fluorescence intensity using the immunochromatographic time-resolved fluorescence test strip and the polystyrene concentration was obtained by fitting the results.

[0050] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and does not limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical means described in the above embodiments or substitute equivalents for some of their technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be within the scope of protection of the present invention.

Claims

1. The device comprises a fluorescent test strip and a sample reaction flask containing a lyophilized anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres. The fluorescent test strip includes a bottom plate, on which an absorbent pad, a detection pad, and a sample pad are attached from top to bottom on the adhesive surface, with adjacent pads overlapping at connection points. The detection pad has a nitrocellulose membrane as its base pad, with a lateral quality control line and a detection line running from top to bottom on the nitrocellulose membrane. The quality control line is coated with an immunoanti-mouse polyclonal antibody, and the detection line is coated with a polystyrene-hemocyanin conjugate. The anti-polystyrene monoclonal antibody is secreted from the hybridoma cell line PS-17, and the deposit number for the hybridoma cell line PS-17 is CCTCC. The item number is C2024145, the deposit date is May 10, 2024, the depositary institution is the China Center for Typical Cultures Depository, and the depositary institution address is located at 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, within the Wuhan University campus. An immunochromatographic time-resolved fluorescence kit for polystyrene micro-nanoplastics, characterized by the following features.

2. The lyophilized product of the anti-polystyrene monoclonal antibody labeled with the aforementioned quantum dot fluorescent microspheres is, Step S11 involves mixing an anti-polystyrene monoclonal antibody and an activated quantum dot fluorescent microsphere labeling reagent in borate buffer, shaking, centrifugation, redissolution, and blocking to obtain an anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres, wherein 1 mL of activated quantum dot fluorescent microsphere labeling reagent is coupled to 40 pg to 90 ug of anti-polystyrene monoclonal antibody. Step S12 involves redissolving the anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres prepared in step S11 in 0.01 mo1 / L, pH 7.4 phosphate buffer containing 1.5% (m / v) trehalose and 2% ( / v) bovine serum albumin, and lyophilizing the mixture in a lyophilizer to obtain a lyophilized product of the anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres. The immunochromatographic time-resolved fluorescence kit for polystyrene micro-nanoplastics as described in claim 1.

3. The method for activating the quantum dot fluorescent microsphere labeling reagent activated in step S11 includes the steps of: dispersing the quantum dot microsphere labeling reagent in pH 8.2, 0.2 M borate buffer by sonication; slowly adding carbodiimide solution; activating by shaking at room temperature; removing the supernatant by centrifugation; redissolving in pH 8.2, 0.2 M borate buffer; and preparing for use, with an activation time of 15 to 30 minutes. The immunochromatographic time-resolved fluorescence kit for polystyrene micro-nanoplastics according to feature 2.

4. The sample reaction flask is a 1-5 mL bayonet flask, and the content of the lyophilized anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres in the sample reaction flask is 100-200 ng. The immunochromatographic time-resolved fluorescence kit for polystyrene micro-nanoplastics as described in claim 1.

5. The aforementioned fluorescent test paper strip is Step S21 involves cutting absorbent paper to obtain an absorbent pad, In the manufacturing step S22 of the detection pad, a coating solution of polystyrene-hemocyanin conjugate is prepared with a coating buffer to a concentration of 0.2 to 0.5 mg / mL, and the coating solution is coated laterally onto the nitrocellulose membrane by line spraying at a position 15 to 20 m away from the upper edge of the nitrocellulose membrane to obtain a detection line. After that, it is dried at 37 to 40°C for 60 to 120 minutes, and the amount of polystyrene-hemocyanin conjugate coating required per 1 cm of detection line is set to 80 to 400 ng. Step S22 involves preparing a coating solution with a concentration of 0.2–0.5 mg / mL using an immunosuppressive mouse polyclonal antibody in a coating buffer, coating a nitrocellulose membrane laterally with the coating solution using a line spray method at a distance of 5–10 m from the detection line to obtain a quality control line, drying the membrane at 37–40°C for 60–120 minutes, and ensuring that the coating amount of immunosuppressive mouse polyclonal antibody per 1 cm of the quality control line is 100–300 ng. Step S23 of the manufacturing of the sample pad, wherein a glass fiber film is wetted in a blocking solution, removed, dried for 10 to 16 hours under conditions of 37 to 40°C to obtain the sample pad, and then stored in a drying oven at room temperature, and Step S24 of the assembly of a fluorescent test paper strip is to attach an absorbent pad, a detection pad, and a sample pad to one side of a cardboard sheet in order from top to bottom, and connect adjacent pads by overlapping them at connection points, with an overlap length of 1 to 3 mm, thereby obtaining a fluorescent test paper strip. The immunochromatographic time-resolved fluorescence kit for polystyrene micro-nanoplastics as described in claim 1.

6. The absorbent pad in the fluorescent test strip has a length of 16 to 18 mm and a width of 3 to 4 mm, the detection pad has a length of 18 to 30 mm and a width of 3 to 4 mm, the sample pad has a length of 10 to 12 mm and a width of 3 to 4 mm, the distance between the detection line on the detection pad in the fluorescent test strip and the upper edge of the nitrocellulose film is 15 to 20 mm, and the distance between the quality control line and the detection line is 5 to 10 mm. The immunochromatographic time-resolved fluorescence kit for polystyrene micro-nanoplastics according to feature 5.

7. The coating buffer for the polystyrene-hemocyanin conjugate in step S22 is prepared by adding water to 1 g of bovine serum albumin, 0.02 g of sodium azide, 0.8 g of sodium chloride, 0.29 g of disodium hydrogen phosphate dodecyl, 0.02 g of potassium chloride, and 0.02 g of potassium dihydrogen phosphate to a final volume of 100 mL. The coating buffer for immunoanti-mouse polyclonal antibodies is prepared by adding water to 0.02 g of sodium azide, 0.8 g of sodium chloride, 0.29 g of disodium hydrogen dodecyl phosphate, 0.02 g of potassium chloride, and 0.02 g of potassium dihydrogen phosphate to a final volume of 100 ml. The immunochromatographic time-resolved fluorescence kit for polystyrene micro-nanoplastics according to feature 5.

8. The blocking solution in step S23 is prepared by adding water to 2 g of ovalbumin, 2 g of sucrose, 0.02 g of sodium azide, 0.8 g of sodium chloride, 0.29 g of disodium hydrogen phosphate dodecyl, 0.02 g of potassium chloride, 0.02 g of potassium dihydrogen phosphate, and 0.5 g of Zeen-20 to a final volume of 100 mL. The immunochromatographic time-resolved fluorescence kit for polystyrene micro-nanoplastics according to feature 5.

9. The kit further includes a sample diluent and a sample diluent pipette, wherein the sample diluent is a Zeen-20 aqueous solution with a volume fraction of 0.01 to 0.30%. The immunochromatographic time-resolved fluorescence kit for polystyrene micro-nanoplastics as described in claim 1.

10. Used to detect the content of polystyrene micro-nanoplastics, Use of an immunochromatographic time-resolved fluorescence kit for polystyrene micro-nanoplastics according to any one of claims 1 to 9.

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