Polystyrene micro-nano plastic immunochromatography time-resolved fluorescence kit and application thereof

Abstract

The invention discloses a polystyrene micro-nano plastic immunochromatography time-resolved fluorescence kit and application thereof.The kit comprises a fluorescent test strip and a sample reaction bottle containing a quantum dot fluorescent microsphere labeled anti-polystyrene monoclonal antibody freeze-dried product, and the fluorescent test strip comprises a bottom plate; a water absorption pad, a detection pad and a sample pad are sequentially adhered to the adhesive surface of the bottom plate from top to bottom, adjacent pads are overlapped and connected at joints, a nitrocellulose membrane serves as a base pad of the detection pad, a transverse quality control line and a detection line are arranged on the nitrocellulose membrane from top to bottom, the quality control line is coated with an anti-mouse-free polyclonal antibody, and the detection line is coated with an anti-mouse-free polyclonal antibody. And the detection line is coated with a polystyrene-hemocyanin conjugate. The immunochromatography time-resolved fluorescence kit provided by the invention can realize quantitative detection of polystyrene, and has the advantages of strong specificity, high sensitivity, simple sample pretreatment, small pollution hazard to the environment, suitability for on-site batch detection and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and particularly relates to a polystyrene micro-nano plastic immunochromatographic time-resolved fluorescence kit and its application. Background Art

[0002] Microplastics are a new type of environmental pollutant that can adsorb heavy metals and persistent organic pollutants and migrate and transform in the environment. Microplastics are easily ingested by animals and release the toxic and harmful substances they carry, causing damage to the growth, development, reproduction, gene expression, etc. of organisms.

[0003] Currently, the main quantitative analysis methods for microplastics include microscopic infrared spectroscopy, microscopic Raman spectroscopy, visual inspection method, etc. The visual inspection method is to count the number of microplastic particles manually, pick out all microplastics and weigh them. It is not only time-consuming and laborious, but also prone to errors in operation. The microscopic infrared spectroscopy and microscopic Raman spectroscopy are basically the same as the visual inspection method in terms of quantification method, but use infrared spectroscopy and Raman spectroscopy to replace the naked eye to identify particles, greatly improving the accuracy of analysis. Infrared and Raman spectroscopy must identify microplastics one by one, and the surface of the identified microplastics cannot be contaminated by organic pollutants, which requires good pretreatment technology and a large amount of analysis time.

[0004] Therefore, a polystyrene micro-nano plastic immunochromatographic time-resolved fluorescence kit and its application are urgently needed to be proposed. Summary of the Invention

[0005] To solve the defects existing in the prior art, the present invention provides a polystyrene micro-nano plastic immunochromatographic time-resolved fluorescence kit and its application.

[0006] To solve the above technical problems, the present invention provides the following technical solutions:

[0007] The first object of the present invention is to provide a polystyrene micro-nano plastic immunochromatographic time-resolved fluorescence kit, which is characterized in that it includes a fluorescence test strip and a sample reaction bottle containing a freeze-dried product of an anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres. The fluorescence test strip includes a bottom plate, and an absorbent pad, a detection pad, and a sample pad are sequentially pasted on the adhesive surface of the bottom plate from top to bottom. Adjacent pads are overlapped and connected at the joint. The detection pad uses a nitrocellulose membrane as the base pad. A horizontal quality control line and a detection line are arranged on the nitrocellulose membrane from top to bottom. The quality control line is coated with rabbit anti-mouse polyclonal antibody, and the detection line is coated with a polystyrene-hemocyanin conjugate. The anti-polystyrene monoclonal antibody is secreted by the hybridoma cell line PS-17. The hybridoma cell line PS-17 is classified and named Hybridoma cell line PS-17. The preservation number of the hybridoma cell line PS-17 is CCTCC NO: C2024145, the preservation date is May 10, 2024, and the preservation unit is the China Center for Type Culture Collection. The address of the preservation unit is within Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0008] Preferably, the freeze-dried product of the anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres is prepared by the following steps:

[0009] S11. Mix and oscillate react the anti-polystyrene monoclonal antibody and the activated quantum dot fluorescent microsphere labeling reagent in a boric acid buffer solution, and then obtain the anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres through centrifugation, reconstitution, and blocking steps; wherein, 40 pg to 90 μg of anti-polystyrene monoclonal antibody is conjugated to 1 mL of the activated quantum dot fluorescent microsphere labeling reagent;

[0010] S12. Reconstitute the anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres prepared in step S11 in a 0.01 mo1 / L pH 7.4 phosphate buffer solution containing 1.5% (m / v) trehalose and 2% (v / v) bovine serum albumin, and place it in a freeze dryer for freeze-drying to obtain a freeze-dried product of the anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres.

[0011] Preferably, the activation method of the activated quantum dot fluorescent microsphere labeling reagent in step S11 includes the following steps: Take the quantum dot microsphere labeling reagent, ultrasonically disperse it with a 0.2M boric acid buffer solution at pH 8.2, then slowly add a carbodiimide solution, activate it by oscillating at room temperature, centrifuge to remove the supernatant, and reconstitute it with a 0.2M boric acid buffer solution at pH 8.2 for standby. The activation time is 15 to 30 minutes.

[0012] Preferably, the sample reaction bottle is a 1-5 mL snap-top vial, and the content of the freeze-dried product of the anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres in the sample reaction bottle is 100-200 ng.

[0013] Preferably, the fluorescent test strip is prepared by the following steps:

[0014] S21. Cut the blotting paper to obtain a blotting pad;

[0015] S22. Preparation of the detection pad: Prepare a coating solution with a concentration of 0.2-0.5 mg / m of the polystyrene-hemocyanin conjugate using a coating buffer, and horizontally coat it on the nitrocellulose membrane by a wire spraying method at a position 15-20 m from the upper edge of the nitrocellulose membrane to obtain a detection line, and then dry it at 37-40 °C for 60-120 minutes; wherein, the coating amount of the polystyrene-hemocyanin conjugate required for each centimeter of the detection line is 80-400 ng;

[0016] Prepare a coating solution with a concentration of 0.2-0.5 mg / mL of the rabbit anti-mouse polyclonal antibody using a coating buffer, and horizontally coat it on the nitrocellulose membrane by a wire spraying method at a position 5-10 m from the detection line to obtain a quality control line, and then dry it at 37-40 °C for 60-120 minutes; wherein, the coating amount of the rabbit anti-mouse polyclonal antibody required for each centimeter of the quality control line is 100-300 ng;

[0017] S23. Preparation of the sample pad: Immerse the glass fiber membrane in the blocking solution, take it out, and dry it at 37-40 °C for 10-16 hours to obtain a sample pad, and then store it at room temperature in a desiccator;

[0018] S24. Assembly of the fluorescent test strip: Paste the blotting pad, the detection pad, and the sample pad on one side of the cardboard in sequence from top to bottom, and overlap and connect the adjacent pads at the connection, and the overlap length is 1-3 mm, that is, the fluorescent test strip is obtained.

[0019] Preferably, the blotting pad in the fluorescent test strip is 16-18 mm long and 3-4 mm wide; the detection pad is 18-30 mm long and 3-4 mm wide; the sample pad is 10-12 mm long and 3-4 mm wide; the distance between the detection line on the detection pad and the upper edge of the nitrocellulose membrane in the fluorescent test strip is 15-20 mm, and the distance between the quality control line and the detection line is 5-10 mm.

[0020] Preferably, the coating buffer used for the polystyrene-ovalbumin conjugate in step S22 is: obtained by dissolving 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 dodecahydrate, 0.02 g of potassium chloride, 0.02 g of potassium dihydrogen phosphate, and adding water to a constant volume of 100 mL;

[0021] The coating buffer for the anti-mouse polyclonal antibody-free is prepared by dissolving 0.02 g of sodium azide, 0.8 g of sodium chloride, 0.29 g of disodium hydrogen phosphate dodecahydrate, 0.02 g of potassium chloride, 0.02 g of potassium dihydrogen phosphate in water and making up the volume to 100 ml.

[0022] Preferably, the blocking solution in step S23 is prepared by dissolving 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 dodecahydrate, 0.02 g of potassium chloride, 0.02 g of potassium dihydrogen phosphate, 0.5 g of Tween-20 in water and making up the volume to 100 mL.

[0023] Preferably, the kit further includes a sample diluent and a sample diluent pipette, and the sample diluent is an aqueous solution of Tween-20 with a volume fraction of 0.01 - 0.30%.

[0024] The second object of the present invention provides an application of the immunochromatographic time-resolved fluorescence kit for polystyrene micro-nano plastics, and the kit is used to detect the content of polystyrene micro-nano plastics.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The immunochromatographic time-resolved fluorescence kit provided by the present invention can achieve quantitative detection of PS, and has the advantages of strong specificity, high sensitivity, simple sample pretreatment, low environmental pollution hazard, suitability for on-site batch detection, etc. In particular, it can eliminate the high background in conventional fluorescence determination. It is a very promising analytical method in non-radioimmunoassay and can be applied to the fields of food, environment, medicine, etc.

[0027] Cell preservation:

[0028] The anti-polystyrene monoclonal antibody provided by the present invention is secreted by the hybridoma cell line PS-17. The hybridoma cell line PS-17 was self-screened by the inventors of the present invention. The hybridoma cell line PS-17 is classified and named Hybridoma cell line PS-17. The preservation number of the hybridoma cell line PS-17 is CCTCC NO: C2024145, the preservation date is May 10, 2024, and the preservation unit is the China Center for Type Culture Collection, and the address of the preservation unit is within Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. Brief description of the drawings

[0029] Figure 1 It is a schematic structural diagram of the fluorescence test strip in Example 1 of the present invention;

[0030] Figure 2It is a schematic diagram of SDS-PAGE electrophoresis of PS-BSA and PS-KLH in Example 2 of the present invention. Detailed implementation mode

[0031] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0032] Example 1: Preparation of a polystyrene micro-nano plastic immunochromatographic time-resolved fluorescence kit.

[0033] This example provides a polystyrene micro-nano plastic immunochromatographic time-resolved fluorescence kit, which includes a fluorescence test strip ( Figure 1 ) and a sample reaction bottle containing a freeze-dried product of a monoclonal anti-polystyrene antibody labeled with quantum dot fluorescent microspheres. The fluorescence test strip includes a bottom plate, and a water absorption 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. Adjacent pads are overlapped and connected at the joint. The detection pad 2 uses a nitrocellulose membrane as the base pad. A horizontal quality control line 4 and a detection line 5 are arranged on the nitrocellulose membrane from top to bottom. The quality control line 4 is coated with a rabbit anti-mouse polyclonal antibody, and the detection line 5 is coated with a polystyrene-keyhole limpet hemocyanin conjugate. The monoclonal anti-polystyrene antibody is secreted by the hybridoma cell line PS-17. The hybridoma cell line PS-17 is classified and named Hybridoma cell line PS-17. The preservation number of the hybridoma cell line PS-17 is CCTCC NO: C2024145, the preservation date is May 10, 2024, and the preservation unit is the China Center for Type Culture Collection. The address of the preservation unit is within Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0034] In this example, the freeze-dried product of the monoclonal anti-polystyrene antibody labeled with quantum dot fluorescent microspheres is prepared by the following steps:

[0035] S11. Mix and oscillate the monoclonal anti-polystyrene antibody and the activated quantum dot fluorescent microsphere labeling reagent in a boric acid buffer solution, and then obtain the monoclonal anti-polystyrene antibody labeled with quantum dot fluorescent microspheres through centrifugation, reconstitution, and blocking steps; among them, 40 pg to 90 μg of the monoclonal anti-polystyrene antibody is conjugated to 1 mL of the activated quantum dot fluorescent microsphere labeling reagent. In this example, 40 pg, 20 μg, or 90 μg can be selected;

[0036] The activation method of the activated quantum dot fluorescent microsphere labeling reagent includes the following steps: Take the quantum dot microsphere labeling reagent, ultrasonically disperse it with a 0.2M boric acid buffer solution at pH 8.2, then slowly add a carbodiimide solution, activate it by shaking at room temperature, centrifuge to remove the supernatant, and redissolve it with a 0.2M boric acid buffer solution at pH 8.2 for standby. The activation time is 15 - 30 min, and 15, 20 or 30 min can be selected in this embodiment. The sample reaction bottle is a 1 - 5 mL snap - cap bottle, and 1, 3 or 4 mL can be selected in this embodiment. The content of the freeze - dried product of the anti - polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres in the sample reaction bottle is 100 - 200 ng, and 100, 150 or 200 ng can be selected in this embodiment.

[0037] S12. Redissolve the anti - polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres prepared in step S11 in a 0.01 mo1 / L phosphate buffer solution at pH 7.4 containing 1.5% (m / v) trehalose and 2% (v / v) bovine serum albumin, and place it in a freeze - dryer for freeze - drying to obtain a freeze - dried product of the anti - polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres.

[0038] In this embodiment, the fluorescent test strip is prepared by the following steps:

[0039] S21. Cut the blotting paper to obtain a blotting pad;

[0040] S22. Preparation of the test pad: Prepare a coating solution with a concentration of 0.2 - 0.5 mg / m of the polystyrene - hemocyanin conjugate with a coating buffer solution. In this embodiment, 0.2, 0.3 or 0.5 mg / m can be selected; at a position 15 - 20 m from the upper edge of the nitrocellulose membrane. In this embodiment, 15, 18 or 20 m can be selected; horizontally coat it on the nitrocellulose membrane by line spraying to obtain a test line, and then dry it at 37 - 40 °C for 60 - 120 minutes. In this embodiment, drying at 37 °C for 60 minutes, drying at 38 °C for 80 minutes or drying at 40 °C for 120 minutes can be selected;

[0041] Among them, the coating amount of the polystyrene - hemocyanin conjugate required for each centimeter of the test line is 80 - 400 ng. In this embodiment, 80, 200 or 400 ng can be selected; the coating buffer solution for the polystyrene - hemocyanin conjugate is: obtained by dissolving 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 dodecahydrate, 0.02 g of potassium chloride, 0.02 g of potassium dihydrogen phosphate in water and making up the volume to 100 mL;

[0042] The anti-mouse polyclonal antibody is formulated into a coating solution with a concentration of 0.2 - 0.5 mg / mL using a coating buffer. In this example, 0.2, 0.3, or 0.5 mg / mL can be selected; at a position 5 - 10 m away from the test line. In this example, 5, 18, or 10 m can be selected; it is transversely coated on the nitrocellulose membrane by a wire spraying method to obtain a quality control line, and then dried at 37 - 40 °C for 60 - 120 minutes. In this example, drying at 37 °C for 60 minutes, drying at 38 °C for 80 minutes, or drying at 40 °C for 120 minutes can be selected;

[0043] Among them, the coating amount of the anti-mouse polyclonal antibody required for each centimeter of the quality control line is 100 - 300 ng. In this example, 100, 200, or 300 ng can be selected; the coating buffer for the anti-mouse polyclonal antibody is prepared by dissolving 0.02 g of sodium azide, 0.8 g of sodium chloride, 0.29 g of disodium hydrogen phosphate dodecahydrate, 0.02 g of potassium chloride, 0.02 g of potassium dihydrogen phosphate, and adding water to a volume of 100 ml;

[0044] S23. Preparation of the sample pad: The glass fiber membrane is soaked in the blocking solution, taken out, and dried at 37 - 40 °C for 10 - 16 hours. In this example, drying at 37 °C for 10 hours, drying at 38 °C for 14 hours, or drying at 40 °C for 16 hours can be selected; to obtain the sample pad, and then stored at room temperature in a desiccator; among them, the blocking solution is prepared by dissolving 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 dodecahydrate, 0.02 g of potassium chloride, 0.02 g of potassium dihydrogen phosphate, 0.5 g of Tween - 20, and adding water to a volume of 100 mL;

[0045] S24. Assembly of the fluorescence test strip: The absorbent pad, test pad, and sample pad are sequentially pasted from top to bottom on one side of the cardboard, and the adjacent pads are overlapped and connected at the joint, and the overlap length is 1 - 3 mm. In this example, 1, 2, or 3 mm can be selected, and thus the fluorescence test strip is obtained.

[0046] In this embodiment, the absorbent pad in the fluorescence test strip is 16 - 18 mm in length, and 16, 17 or 18 mm can be selected in this embodiment, 3 - 4 mm in width, and 3, 3.5 or 4 mm can be selected in this embodiment; the detection pad is 18 - 30 mm in length, and 18, 25 or 30 mm can be selected in this embodiment, 3 - 4 mm in width, and 3, 3.5 or 4 mm can be selected in this embodiment; the sample pad is 10 - 12 mm in length, and 10, 11 or 12 mm can be selected in this embodiment, 3 - 4 mm in width, and 3, 3.5 or 4 mm can be selected in this embodiment; the distance between the detection line on the detection pad in the fluorescence test strip and the upper edge of the nitrocellulose membrane is 15 - 20 mm, and 15, 18 or 20 mm can be selected in this embodiment, and the distance between the quality control line and the detection line is 5 - 10 mm, and 5, 6 or 10 mm can be selected in this embodiment.

[0047] In this embodiment, the kit further includes a sample diluent, and the sample diluent is an aqueous solution of Tween - 20 with a volume fraction of 0.01 - 0.30%.

[0048] Example 2: Preparation of anti - polystyrene monoclonal antibody.

[0049] (1) Coupling of micro - plastic (PS - CHOOH) with coupling protein (BSA / KLH): The micro - plastic, coupling buffer solution (Polylink coupling buffer), coupling carbodiimide (Polylink EDAC), and coupling buffer were cooled to room temperature. Take 12.5 mg of micro - plastic into a 1.5 mL centrifuge tube, centrifuge at 500 - 1000 g for 5 - 10 min, and discard the supernatant; add 0.4 mL of coupling buffer solution to resuspend, centrifuge again, and discard the supernatant; add 0.17 mL of coupling buffer solution to resuspend again. Dissolve 10 mg of polylink EDAC in 50 μL of coupling buffer solution to prepare a 200 mg / mL EDAC solution, and note that it should be prepared and used immediately. Add 20 μL of EDAC solution to the 0.17 mL coupling buffer solution suspension, fix it on a rotator and mix well at room temperature for 15 min for activation. Add 200 - 500 μg of coupling protein (BSA / KLH), fix it on a rotator and mix well at room temperature for 30 - 60 min. (Dissolve the protein in coupling buffer solution to 1 - 5 mg / ml). Centrifuge at 500 - 1000 g for 10 min, aspirate the supernatant to detect the amount of coupling protein (BSA / KLH). Add 0.4 mL of coupling wash / storage solution (polylink wash / storage buffer) to resuspend, and the precipitate at the bottom of the tube is PS - BSA or PS - KLH, and finally store it at 4°C. Perform SDS - PAGE analysis (4% stacking gel and 7.5% separating gel). The detection result of reducing SDS - PAGE is as Figure 2 shown, proving the successful coupling of PS - KLH and PS - BSA.

[0050] (2) Preparation of monoclonal antibodies: Female Balb / c mice aged 6 - 8 weeks were selected, and the prepared PS - BSA artificial antigen was used for immunization injection. Adult Balb / c mice received primary immunization by subcutaneous administration (in the area between the shoulders), and booster immunization was carried out every 2 weeks. Blood samples were collected at week 0 (before immunization), week 8 (2 weeks after the fourth booster injection), week 10 (before the third booster injection), week 12, and week 14. Then, serum was separated to obtain B lymphocytes, RNA was extracted and transcribed into a cDNA library. Through cDNA re - amplification, specific gene fragments were obtained and recombined on phages to construct a nanobody gene library. Then, specific antibody screening was carried out to obtain antibodies that met the requirements. The PS - BSA artificial antigen was immobilized on a carrier, and then allowed to interact with the phage library. Then, the surface of the carrier was washed to remove unbound or non - specifically bound phages, and the binding was disrupted with strong acid to obtain a positive phage solution. After repeating 2 - 3 rounds, monoclonal antibodies that met the requirements could be obtained.

[0051] By injecting the PS - BSA artificial antigen, the immunization protocol for mice is shown in Table 1, observing what specific reactions it produces on the immune system of Balb / c mice and exploring the activation mechanism of antibody - specific immune responses.

[0052] Table 1: Immunization protocol for mice.

[0053]

[0054] The obtained monoclonal antibody contains a heavy - chain variable region and a light - chain variable region. Both the heavy - chain variable region and the light - chain variable region are composed of a complementarity - determining region and a framework region; the complementarity - determining regions of both the heavy - chain variable region and the light - chain variable region are composed of CDR1, CDR2, and CDR3;

[0055] The amino acid sequence of CDR1 of the heavy - chain variable region is shown as positions 50 - 54 of SEQ ID No.1;

[0056] The amino acid sequence of CDR2 of the heavy - chain variable region is shown as positions 69 - 85 of SEQ ID No.1;

[0057] The amino acid sequence of CDR3 of the heavy - chain variable region is shown as positions 118 - 128 of SEQ ID No.1;

[0058] The amino acid sequence of CDR1 of the light - chain variable region is shown as positions 44 - 54 of SEQ ID No.2;

[0059] The amino acid sequence of CDR2 of the light - chain variable region is shown as positions 70 - 76 of SEQ ID No.2;

[0060] The amino acid sequence of CDR3 in the light chain variable region is shown as positions 109 - 117 of SEQ ID No.2.

[0061] The amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID No.1:

[0062] MEWIWIFLFILSGTAGVHSQVQLQQSGAELARPGASVKLSCKASGYTFTDYYINWVKQRTGQGLEWIGEIYPGSGNTYYNEKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCARSEIYGIYYFDYWGQGTTLTVSS.

[0063] The amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID No.2:

[0064] METHSQVFVYMLLWLSGVEGDIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPYTFGGGTKLEIK.

[0065] Example 3: Application of a polystyrene micro - nano plastic immunochromatographic time - resolved fluorescence kit.

[0066] The specific principle is as follows: After the sample to be tested is pretreated to obtain the sample solution to be tested, it is added to the sample reaction bottle, mixed evenly, and a fluorescence test strip is inserted. After reacting at 37 °C for 6 - 10 minutes, it is detected with a time - resolved fluorescence tester to obtain the ratio of the fluorescence intensity of the test line (T) to the fluorescence intensity of the control line (C) on the fluorescence immunochromatographic test strip; based on the relationship curve between the ratio of the fluorescence intensity of the test line and the fluorescence intensity of the control line on the fluorescence immunochromatographic test strip and the polystyrene concentration, the content of polystyrene in the sample solution to be tested is obtained, and finally, through conversion, the content of polystyrene in the sample to be tested is obtained.

[0067] Water samples and soil samples are collected from oceans, rivers, lakes and waters. Textile wastewater samples are collected from textile enterprises. Food samples are collected from fields or dining tables. The collected samples are quantitatively detected by a reagent strip after pretreatment and extraction.

[0068] The pretreatment is carried out according to the following steps:

[0069] (1) For liquid samples such as water samples, each sample is centrifuged at 17000×g for 3 minutes, and then the supernatant is subjected to digestion and extraction. The digestion and extraction solution is diluted with a sample diluent and used for quantitative immunochromatographic detection with the test strip.

[0070] (2) For non-liquid samples, the samples need to be digested, microplastics extracted, and diluted with diluent before immunochromatographic quantitative detection.

[0071] Taking soil samples (spiked detection) as an example: Deionized water was added to the soil samples (1:1 w / v), and then vortexed for 10 s. The slurry mixture was centrifuged at 17000×g for 3 minutes, and the supernatant was used as a specific matrix extract for spiked recovery analysis. Briefly, 50 μL of polystyrene (25 μg) was added to 950 μL of matrix or matrix extract, mixed and incubated at 37° for 16 hours. After incubation, the samples were centrifuged at 5500 rpm for 3 minutes, and the supernatant was discarded. The polystyrene particles were suspended and washed four times with PBST washing buffer (each wash was incubated for 3 minutes). All assays were performed using duplicate matrix samples. The washing solution PBST was used as a control matrix. Subsequently, the washed polystyrene particles were subjected to the same immunochromatographic assay as described above.

[0072] According to the above protocol, the relationship curve between the ratio (T / C) of the fluorescence intensity of the test line (T) to the fluorescence intensity of the control line (C) of the fluorescence test strip and the polystyrene concentration was obtained by the following method:

[0073] (1) A series of polystyrene standard solutions with different concentrations were prepared;

[0074] (2) Appropriate amounts of the above polystyrene standard solutions with different concentrations were respectively added to the sample reaction bottles, mixed well, inserted with fluorescence test strips, and reacted at 37 °C for 6 - 10 minutes. The time-resolved fluorescence intensities of the test line (T) and the control line of each immunochromatographic time-resolved fluorescence test strip were detected with a time-resolved fluorescence immunoassay analyzer, and thus the ratio (T / C) of the fluorescence intensity of the test line to the fluorescence intensity of the control line of each immunochromatographic time-resolved fluorescence test strip was obtained. The relationship curve between the ratio of the fluorescence intensity of the test line to the fluorescence intensity of the control line of the immunochromatographic time-resolved fluorescence test strip and the polystyrene concentration was obtained by fitting.

[0075] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A polystyrene micro-nano plastic immunochromatographic time-resolved fluorescence kit, characterized in that, It includes a fluorescent test strip and a sample reaction bottle containing a freeze-dried product of anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres. The fluorescent test strip includes a bottom plate. The adhesive surface of the bottom plate is successively pasted with a water-absorbing pad, a detection pad, and a sample pad from top to bottom. Adjacent pads are overlapped and connected at the joint. The detection pad uses a nitrocellulose membrane as the base pad. A horizontal quality control line and a detection line are arranged on the nitrocellulose membrane from top to bottom. The quality control line is coated with rabbit anti-mouse polyclonal antibody, and the detection line is coated with a polystyrene-hemocyanin conjugate. The anti-polystyrene monoclonal antibody is secreted by the hybridoma cell line PS-17. The hybridoma cell line PS-17 is classified and named Hybridoma cell line PS-17. The preservation number of the hybridoma cell line PS-17 is CCTCC NO:C2024145, the preservation date is May 10, 2024, and the preservation unit is the China Center for Type Culture Collection, and the address of the preservation unit is within Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. The immunochromatographic time-resolved fluorescence kit for polystyrene micro-nano plastics according to claim 1, characterized in that, The freeze-dried product of anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres is prepared by the following steps: S11. Mix and oscillate react the anti-polystyrene monoclonal antibody and the activated quantum dot fluorescent microsphere labeling reagent in a boric acid buffer solution, and then obtain the anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres through centrifugation, reconstitution, and blocking steps; among them, 40 pg to 90 μg of anti-polystyrene monoclonal antibody is conjugated to 1 mL of the activated quantum dot fluorescent microsphere labeling reagent; S12. Reconstitute the anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres prepared in step S11 in a 0.01 mo1 / L pH 7.4 phosphate buffer solution containing 1.5% (m / v) trehalose and 2% (v / v) bovine serum albumin, and place it in a freeze dryer for freeze-drying to obtain the freeze-dried product of anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres.

3. The immunochromatographic time-resolved fluorescence kit for polystyrene micro-nano plastics according to claim 2, wherein The activation method of the activated quantum dot fluorescent microsphere labeling reagent in step S11 includes the following steps: Take the quantum dot microsphere labeling reagent, ultrasonically disperse it with a 0.2M boric acid buffer solution at pH 8.2, then slowly add a carbodiimide solution, activate it by shaking at room temperature, centrifuge to remove the supernatant, and reconstitute it with a 0.2M boric acid buffer solution at pH 8.2 for standby. The activation time is 15 to 30 minutes.

4. A polystyrene micro-nano plastic immunochromatographic time-resolved fluorescence kit according to claim 1, wherein The sample reaction bottle is a 1-5 mL snap-top bottle, and the content of the freeze-dried product of anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres in the sample reaction bottle is 100 to 200 ng.

5. A polystyrene micro-nano plastic immunochromatographic time-resolved fluorescence kit according to claim 1, characterized in that, The fluorescent test strip is prepared by the following steps: S21. Cut the absorbent paper to obtain the water-absorbing pad; S22. Preparation of the detection pad: Prepare a coating solution with a concentration of 0.2 - 0.5 mg / m of the polystyrene - hemocyanin conjugate using a coating buffer. At a position 15 - 20 m from the upper edge of the nitrocellulose membrane, transversely coat it on the nitrocellulose membrane by the wire spraying method to obtain a detection line, and then dry it at 37 - 40 °C for 60 - 120 minutes; among them, the coating amount of the polystyrene - hemocyanin conjugate required for each centimeter of the detection line is 80 - 400 ng; Prepare a coating solution with a concentration of 0.2 - 0.5 mg / mL of the rabbit anti - mouse polyclonal antibody using a coating buffer. At a position 5 - 10 m from the detection line, transversely coat it on the nitrocellulose membrane by the wire spraying method to obtain a quality control line, and then dry it at 37 - 40 °C for 60 - 120 minutes; among them, the coating amount of the rabbit anti - mouse polyclonal antibody required for each centimeter of the quality control line is 100 - 300 ng S23. Preparation of the sample pad: Immerse the glass fiber membrane in the blocking solution, take it out, and dry it at 37 - 40 °C for 10 - 16 hours to obtain the sample pad, and then store it at room temperature in a desiccator; S24. Assembly of the fluorescence test strip: Paste the absorbent pad, detection pad, and sample pad on one side of the cardboard from top to bottom in sequence. The adjacent pads are overlapped and connected at the joint, and the overlapping length is 1 - 3 mm, thus obtaining the fluorescence test strip.

6. The immunochromatographic time-resolved fluorescence kit for polystyrene micro-nano plastics according to claim 5, wherein In the fluorescence test strip, the absorbent pad is 16 - 18 mm long and 3 - 4 mm wide; the detection pad is 18 - 30 mm long and 3 - 4 mm wide; the sample pad is 10 - 12 mm long and 3 - 4 mm wide; the distance between the detection line on the detection pad and the upper edge of the nitrocellulose membrane in the fluorescence test strip is 15 - 20 mm, and the distance between the quality control line and the detection line is 5 - 10 mm.

7. A polystyrene micro-nano plastic immunochromatographic time-resolved fluorescence kit according to claim 5, characterized in that, The coating buffer used for the polystyrene - hemocyanin conjugate in step S22 is: Prepared by dissolving 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 dodecahydrate, 0.02 g of potassium chloride, 0.02 g of potassium dihydrogen phosphate in water and making up the volume to 100 mL; The coating buffer for the rabbit anti - mouse polyclonal antibody is: Prepared by dissolving 0.02 g of sodium azide, 0.8 g of sodium chloride, 0.29 g of disodium hydrogen phosphate dodecahydrate, 0.02 g of potassium chloride, 0.02 g of potassium dihydrogen phosphate in water and making up the volume to 100 ml.

8. A polystyrene micro-nano plastic immunochromatographic time-resolved fluorescence kit according to claim 5, characterized in that, The blocking solution in step S23 is: Prepared by dissolving 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 dodecahydrate, 0.02 g of potassium chloride, 0.02 g of potassium dihydrogen phosphate, 0.5 g of Tween - 20 in water and making up the volume to 100 mL.

9. A polystyrene micro-nano plastic immunochromatographic time-resolved fluorescence kit according to claim 1, characterized in that, The kit also includes a sample diluent and a sample diluent pipette, and the sample diluent is an aqueous solution of Tween - 20 with a volume fraction of 0.01 - 0.30%.

10. Application of a polystyrene micro - nano plastic immunochromatographic time - resolved fluorescence kit as described in any one of claims 1 - 9, using the kit to detect the content of polystyrene micro - nano plastics.

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