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

Abstract

The application discloses a polystyrene micro-nano plastic immunochromatography time-resolved fluorescence kit and application thereof. The kit comprises a fluorescence test strip and a sample reaction bottle containing quantum dot fluorescence microsphere labeled anti-polystyrene monoclonal antibody freeze-dried product. The fluorescence test strip comprises a bottom plate, the adhesive surface of the bottom plate is sequentially pasted with a water absorption pad, a detection pad and a sample pad from top to bottom, adjacent pads are connected through overlapping connection at a connecting position, the detection pad takes a nitrocellulose membrane as a base 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 anti-mouse polyclonal antibody, and a polystyrene-hemocyanin conjugate is coated on the detection line. The immunochromatography time-resolved fluorescence kit provided by the application can realize quantitative detection of polystyrene, has the advantages of high specificity, high sensitivity, simple sample pretreatment, small environmental pollution hazard and suitability for on-site batch detection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological materials, and particularly relates to a polystyrene micro-nano plastic immunochromatography time-resolved fluorescence kit and application thereof. BACKGROUND

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

[0003] At present, the quantitative analysis methods for microplastics mainly include microscopic infrared spectroscopy, microscopic Raman spectroscopy and visual method. The visual method is a manual counting method, which counts the number of microplastic particles and weighs all the microplastics. It is not only time-consuming and laborious, but also prone to errors in operation. Microscopic infrared spectroscopy and microscopic Raman spectroscopy are basically consistent with visual method in quantitative method, but infrared spectroscopy and Raman spectroscopy are used instead of naked eye identification of particles, which greatly improves the accuracy of analysis. Infrared and Raman spectroscopy must identify microplastics one by one, and the identified microplastic surface 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 immunochromatography time-resolved fluorescence kit and application thereof are urgently needed. SUMMARY

[0005] In order to solve the defects in the prior art, the application provides a polystyrene micro-nano plastic immunochromatography time-resolved fluorescence kit and application thereof.

[0006] In order to solve the above technical problems, the application provides the following technical scheme:

[0007] The first object of the application provides a polystyrene micro-nano plastic immunochromatographic time-resolved fluorescence kit, characterized by comprising a fluorescence test strip and a sample reaction bottle containing quantum dot fluorescence microsphere labeled anti-polystyrene monoclonal antibody freeze-dried product, wherein the fluorescence test strip comprises a bottom plate, the adhesive surface of the bottom plate is sequentially pasted with a water absorption pad, a detection pad and a sample pad from top to bottom, and adjacent pads are connected by overlapping at the connection part, the detection pad takes nitrocellulose membrane as a base 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 anti-mouse polyclonal antibody, and the detection line is coated with polystyrene-hemocyanin conjugate, the anti-polystyrene monoclonal antibody is secreted by a hybridoma cell strain PS-17, the hybridoma cell strain PS-17 is classified and named as Hybridoma cell line PS-17, the preservation number of the hybridoma cell strain PS-17 is CCTCC NO: C2024145, the preservation date is May 10, 2024, and the preservation unit is China Center for Type Culture Collection, Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province.

[0008] Preferably, the quantum dot fluorescence microsphere labeled anti-polystyrene monoclonal antibody freeze-dried product is prepared by the following steps:

[0009] S11, mixing, oscillating and reacting anti-polystyrene monoclonal antibody and activated quantum dot fluorescence microsphere labeled reagent in boric acid buffer, and then obtaining quantum dot fluorescence microsphere labeled anti-polystyrene monoclonal antibody through centrifugation, reconstitution and blocking steps; wherein 1 mL of activated quantum dot fluorescence microsphere labeled reagent is coupled with 40 pg to 90 ug of anti-polystyrene monoclonal antibody;

[0010] S12, reconstituting the quantum dot fluorescence microsphere labeled anti-polystyrene monoclonal antibody prepared in step S11 in 0.01 mol / L pH 7.4 phosphate buffer containing 1.5% m / v trehalose and 2% m / v bovine serum albumin, and freeze-drying in a freeze dryer to obtain quantum dot fluorescence microsphere labeled anti-polystyrene monoclonal antibody freeze-dried product.

[0011] Preferably, the activation method of the activated quantum dot fluorescence microsphere labeled reagent in step S11 comprises the following steps: taking quantum dot microsphere labeled reagent, ultrasonic dispersing in pH 8.2 0.2M boric acid buffer, then slowly adding carbodiimide solution, oscillating at room temperature for activation, centrifuging to remove supernatant, reconstituting in pH 8.2 0.2M boric acid buffer, and standby, and the activation time is 15 to 30 minutes.

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

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

[0014] S21, cutting the absorbent paper into an absorbent pad;

[0015] S22, preparation of the detection pad: the polystyrene-hemocyanin conjugate is prepared into a coating solution with a concentration of 0.2-0.5 mg / mL using a coating buffer, and is coated on the nitrocellulose membrane in a line-jet manner at a position 15-20 mm away from the edge of the nitrocellulose membrane to obtain a detection line, and then dried at 37-40°C for 60-120 minutes; wherein the coating amount of the polystyrene-hemocyanin conjugate required per centimeter of the detection line is 80-400 ng;

[0016] The anti-mouse polyclonal antibody is prepared into a coating solution with a concentration of 0.2-0.5 mg / mL using a coating buffer, and is coated on the nitrocellulose membrane in a line-jet manner at a position 5-10 mm away from the detection line to obtain a quality control line, and then dried at 37-40°C for 60-120 minutes; wherein the coating amount of the anti-mouse polyclonal antibody required per centimeter of the quality control line is 100-300 ng;

[0017] 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 to obtain a sample pad, and then stored in a desiccator at room temperature;

[0018] S24, assembly of the fluorescent test strip: the absorbent pad, the detection pad, and the sample pad are sequentially pasted on one side of the paperboard from top to bottom, and the adjacent pads are connected by overlapping at the connection part with an overlapping length of 1-3 mm, thereby obtaining the fluorescent test strip.

[0019] Preferably, the length of the absorbent pad in the fluorescent test strip is 16-18 mm, and the width is 3-4 mm; the length of the detection pad is 18-30 mm, and the width is 3-4 mm; the length of the sample pad is 10-12 mm, and the width is 3-4 mm; the distance between the detection line on the detection pad and the 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 for the polystyrene-ovalbumin 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 dodecahydrate, 0.02 g of potassium chloride, 0.02 g of potassium dihydrogen phosphate, and 100 mL of water;

[0021] The coating buffer for the anti-mouse polyclonal antibody is: 0.02 g sodium azide, 0.8 g sodium chloride, 0.29 g disodium hydrogen phosphate dodecahydrate, 0.02 g potassium chloride, 0.02 g potassium dihydrogen phosphate, and water to 100 ml.

[0022] Preferably, the blocking solution in the step S23 is: 2 g ovalbumin, 2 g sucrose, 0.02 g sodium azide, 0.8 g sodium chloride, 0.29 g disodium hydrogen phosphate dodecahydrate, 0.02 g potassium chloride, 0.02 g potassium dihydrogen phosphate, 0.5 g Tween-20, and water to 100 ml.

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

[0024] The second object of the present application provides a non-diagnostic application of the polystyrene micro-nano plastic immunochromatographic time-resolved fluorescence kit, and the kit is used for detecting the content of polystyrene micro-nano plastic.

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

[0026] The immunochromatographic time-resolved fluorescence kit provided by the present application can realize quantitative detection of PS, has the advantages of strong specificity, high sensitivity, simple sample pretreatment, small environmental pollution hazard, suitability for on-site batch detection, etc., especially can eliminate the high background in conventional fluorescence determination, is a very potential analysis method in non-radioimmunoassay, and can be applied to the fields of food, environment, medical treatment, etc.

[0027] Cell preservation:

[0028] The anti-polystyrene monoclonal antibody provided by the present application is secreted by the hybridoma cell strain PS-17, the hybridoma cell strain PS-17 is screened by the inventors of the present application, the classification name of the hybridoma cell strain PS-17 is Hybridoma cell line PS-17, the preservation number of the hybridoma cell strain PS-17 is CCTCC NO: C2024145, the preservation date is May 10, 2024, the preservation unit is China Center for Type Culture Collection, and the address of the preservation unit is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 is a structural schematic diagram of the fluorescence test strip in embodiment 1 of the present application.

[0030] Figure 2Figure 1 is a schematic diagram of SDS-PAGE electrophoresis of PS-BSA and PS-KLH in Example 2 of the present application. DETAILED DESCRIPTION

[0031] The preferred embodiments of the present application are described below in conjunction with the accompanying drawings, it should be understood that the preferred embodiments described herein are only used to illustrate and explain the present application, and are not used to limit the present application.

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

[0033] The present embodiment provides a polystyrene micro-nano plastic immunochromatographic time-resolved fluorescence kit, which comprises a fluorescence test strip (1) and a sample reaction bottle containing quantum dot fluorescent microsphere labeled anti-polystyrene monoclonal antibody freeze-dried product. Figure 1 The fluorescence test strip comprises a bottom plate, and the adhesive surface of the bottom plate is sequentially pasted with a water absorption pad 1, a detection pad 2 and a sample pad 3 from top to bottom, and adjacent pads are connected by overlapping at the connection. The detection pad 2 uses nitrocellulose membrane as the base pad, and the nitrocellulose membrane is provided with a lateral quality control line 4 and a detection line 5 from top to bottom. The quality control line 4 is coated with anti-mouse polyclonal antibody, and the detection line 5 is coated with polystyrene-hemocyanin conjugate. The anti-polystyrene monoclonal antibody is secreted by a hybridoma cell strain PS-17, the classification name of the hybridoma cell strain PS-17 is Hybridoma cell line PS-17, the preservation number of the hybridoma cell strain PS-17 is CCTCC NO: C2024145, the preservation date is May 10, 2024, and the preservation unit is China Center for Type Culture Collection, Wuhan University, Wuhan, Hubei, China.

[0034] In the present embodiment, the quantum dot fluorescent microsphere labeled anti-polystyrene monoclonal antibody freeze-dried product is prepared by the following steps:

[0035] S11, mix and oscillate the anti-polystyrene monoclonal antibody and the activated quantum dot fluorescent microsphere labeling reagent in boric acid buffer, then centrifuge, reconstitute and block to obtain the quantum dot fluorescent microsphere labeled anti-polystyrene monoclonal antibody; wherein 1 mL of activated quantum dot fluorescent microsphere labeling reagent is coupled with 40 pg to 90 ug of anti-polystyrene monoclonal antibody, and in the present embodiment, 40 pg, 20 ug or 90 ug can be selected.

[0036] The activation method of the activated quantum dot fluorescent microsphere labeling reagent comprises the following steps: taking the quantum dot microsphere labeling reagent, ultrasonic dispersion in a 0.2M boric acid buffer solution with pH 8.2, then slowly adding a carbodiimide solution, room temperature vibration activation, centrifugal supernatant, re-dissolving in a 0.2M boric acid buffer solution with pH 8.2, standby, the activation time is 15-30min, in this embodiment, 15, 20 or 30min can be selected. The sample reaction bottle is a 1-5mL bayonet bottle, in this embodiment, 1, 3 or 4mL can be selected, and the content of the quantum dot fluorescent microsphere labeled anti-polystyrene monoclonal antibody freeze-dried product in the sample reaction bottle is 100-200ng, in this embodiment, 100, 150 or 200ng can be selected.

[0037] S12, re-dissolving the quantum dot fluorescent microsphere labeled anti-polystyrene monoclonal antibody prepared in step S11 in a 0.01mol / L pH 7.4 phosphate buffer solution containing 1.5% m / v trehalose and 2% m / v bovine serum albumin, and freeze-drying in a freeze dryer to obtain a quantum dot fluorescent microsphere labeled anti-polystyrene monoclonal antibody freeze-dried product.

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

[0039] S21, cutting the water-absorbing paper into a water-absorbing pad;

[0040] S22, preparation of the detection pad: preparing the polystyrene-hemocyanin conjugate into a coating solution with a concentration of 0.2-0.5mg / m in a coating buffer, in this embodiment, 0.2, 0.3 or 0.5mg / m can be selected; coating it transversely on the nitrocellulose membrane at a position 15-20mm away from the nitrocellulose membrane, in this embodiment, 15, 18 or 20mm can be selected; using a line spraying method to obtain a detection line, and then drying at 37-40℃ for 60-120min, in this embodiment, 37℃ for 60min, 38℃ for 80min or 40℃ for 120min can be selected;

[0041] In this embodiment, 80, 200 or 400ng can be selected; the coating buffer for the polystyrene-hemocyanin conjugate is: adding 1g bovine serum albumin, 0.02g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate dodecahydrate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate, and water to make up to 100mL;

[0042] The anti-mouse polyclonal antibody was prepared with coating buffer to a concentration of 0.2-0.5 mg / mL. In this embodiment, 0.2, 0.3, or 0.5 mg / mL can be selected. The antibody was coated laterally onto the nitrocellulose membrane at a distance of 5-10 mm from the detection line. In this embodiment, 5, 18, or 10 mm can be selected. This was used to obtain the control line. The membrane was then dried at 37-40°C for 60-120 minutes. In this embodiment, drying at 37°C for 60 minutes, at 38°C for 80 minutes, or at 40°C for 120 minutes can be selected.

[0043] The amount of anti-mouse polyclonal antibody required for coating each centimeter of quality control line is 100~300ng, and in this embodiment, 100, 200 or 300ng can be selected; the coating buffer for anti-mouse polyclonal antibody is: 0.02g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate dodecahydrate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate, and water are added to a final volume of 100ml;

[0044] S23. Preparation of sample pad: Immerse the glass fiber membrane in the blocking solution, remove it, and dry it at 37~40℃ for 10~16 hours. In this embodiment, drying at 37℃ for 10 hours, 38℃ for 14 hours, or 40℃ for 16 hours can be selected. The sample pad is then stored in a desiccator at room temperature. The blocking solution is prepared by adding 2g ovalbumin, 2g sucrose, 0.02g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate dodecahydrate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate, and 0.5g Tween-20 to water to a final volume of 100mL.

[0045] S24. Assembly of fluorescent test strips: On one side of the cardboard, the absorbent pad, the detection pad, and the sample pad are pasted sequentially from top to bottom. Adjacent pads are overlapped at the joints, with an overlap length of 1 to 3 mm. In this embodiment, 1, 2, or 3 mm can be selected to obtain the fluorescent test strip.

[0046] In the present embodiment, the water absorption pad in the fluorescent test strip is 16-18 mm long, and in the present embodiment, 16, 17 or 18 mm can be selected, 3-4 mm wide, and in the present embodiment, 3, 3.5 or 4 mm can be selected; the detection pad is 18-30 mm long, and in the present embodiment, 18, 25 or 30 mm can be selected, 3-4 mm wide, and in the present embodiment, 3, 3.5 or 4 mm can be selected; the sample pad is 10-12 mm long, and in the present embodiment, 10, 11 or 12 mm can be selected, 3-4 mm wide, and in the present embodiment, 3, 3.5 or 4 mm can be selected; the distance between the detection line on the detection pad and the nitrocellulose membrane in the fluorescent test strip is 15-20 mm, and in the present embodiment, 15, 18 or 20 mm can be selected, and the distance between the quality control line and the detection line is 5-10 mm, and in the present embodiment, 5, 6 or 10 mm can be selected.

[0047] In the present embodiment, the kit further comprises a sample diluent, which is a 0.01-0.30% (volume fraction) Tween-20 aqueous solution.

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

[0049] (1) Coupling of microplastics (PS-CHOOH) and coupling proteins (BSA / KLH): Cool the coupling buffer solution (Polylink coupling buffer) and the coupling carbodiimide (Polylink EDAC) to room temperature. Take 12.5 mg of microplastics 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, which should be prepared fresh. Add 20 μL of the EDAC solution to the 0.17 mL of coupling buffer solution suspension, and mix on a rotator at room temperature for 15 min for activation. Add 200-500 μg of coupling protein (BSA / KLH), and mix on a rotator 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, and remove 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, which is finally stored at 4°C. Perform SDS-PAGE analysis (4% concentrated gel and 7.5% separation gel). The results of reduced SDS-PAGE detection are as follows: Figure 2As shown, it is proved that the PS-KLH and PS-BSA coupling is successful.

[0050] (2) Preparation of monoclonal antibody: 6-8 weeks old female Balb / c mice are selected, and the prepared PS-BSA artificial antigen is immunized. Adult Balb / c mice are given primary immunization by subcutaneous injection (in the area between the shoulders), and are given booster immunization every 2 weeks. Blood samples are collected at 0 weeks (before immunization), 8 weeks (2 weeks after the fourth booster injection), 10 weeks (before the third booster injection), 12 weeks and 14 weeks, and then serum separation is performed to obtain B lymphocytes, RNA is extracted and transcribed into a cDNA library. Specific gene fragments are obtained by re-amplification of cDNA, and are recombined on phages to construct a nanobody gene library. Then specific antibody screening is performed to obtain the required antibody, and the PS-BSA artificial antigen is fixed on the carrier, and then interacts with the phage library. Then the carrier surface is washed to wash away the unbound or non-specifically bound phages, and then the binding is destroyed by strong acid, so that the positive phage solution is obtained. After 2-3 rounds of repetition, the required monoclonal antibody is obtained.

[0051] By injecting the PS-BSA artificial antigen, the immunization scheme of the mice is shown in Table 1, and the specific response of the immune system of the Balb / c mice is observed, and the activation mechanism of the antibody specific immune response is explored.

[0052] Table 1: Immunization scheme of mice.

[0053]

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

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

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

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

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

[0059] The amino acid sequence of CDR2 of the heavy chain variable region is shown as positions 70-76 of SEQ ID No. 1.

[0060] The amino acid sequence of CDR3 of the heavy chain variable region is shown as positions 115-123 of SEQ ID No. 1.

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

[0062] MEWIWIFLFILSGTAGVHSQVQLQQSGAELARPGASVKLSCKASGYTFTDYYINWVKQRTGQGLEWIGEIYPGSGNTYYNEKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCARSEIYGIYYFDYWGQGTTLTVSS.

[0063] The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID No. 1:

[0064] METHSQVFVYMLLWLSGVEGDIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPYTFGGGTKLEIK.

[0065] Example 3: Application of polystyrene micro-nano plastic immunochromatographic time-resolved fluorescence kit for non-diagnostic purposes.

[0066] The specific principle is that after the sample to be tested is pretreated to obtain a sample solution to be tested, the sample solution is added to a sample reaction bottle, mixed, inserted into a fluorescence test strip, and reacted at 37℃ for 6-10 minutes, then detected by a time-resolved fluorescence tester to obtain the ratio of the fluorescence intensity of the detection line (T) to the fluorescence intensity of the quality control line (C) on the fluorescence immunochromatographic 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 of the fluorescence immunochromatographic test strip and the polystyrene concentration, the content of polystyrene in the sample solution to be tested is obtained, and finally the content of polystyrene in the sample to be tested is obtained after conversion.

[0067] Water samples, soil samples are collected from the sea, rivers and lakes. Textile wastewater samples are collected from textile enterprises. Food samples are collected from the field or the table. After pretreatment, the samples are extracted and detected by the reagent strip.

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

[0069] (1) Liquid samples, such as water samples, etc. will be centrifuged at 17000xg for 3 minutes for each sample, and then the supernatant will be digested and extracted. The digested and extracted solution will be diluted with sample diluent and then used for quantitative detection by test strip immunochromatography.

[0070] (2) For non-liquid samples, the samples need to be digested, microplastic extracted, diluted with diluent, and then subjected to quantitative detection by immunochromatography.

[0071] Take soil samples (spiked detection) as an example: Add deionized water to the soil sample (1:1 w / v), then vortex for 10 s. Centrifuge the slurry mixture at 17000xg for 3 minutes, and use the supernatant as a specific matrix extract for spiked recovery analysis. Briefly, 50µL of polystyrene (25µg) was added to 950µL 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 wash buffer (3 minutes incubation per wash). All assays used duplicate matrix samples. The wash 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 scheme, the relationship curve between the ratio of the fluorescence intensity of the test line (T) to the fluorescence intensity of the quality control line (C) of the fluorescent test strip and the polystyrene concentration is obtained by the following method:

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

[0074] (2) An appropriate amount of polystyrene standard solution of each concentration is added to the sample reaction bottle, mixed, inserted into the fluorescent test strip, and incubated at 37°C for 6-10 minutes. The time-resolved fluorescence intensity (C) of the test line (T) and the quality control line of each immunochromatographic time-resolved fluorescence test strip is detected by a time-resolved fluorescence immunoassay instrument, and the ratio of the fluorescence intensity of the test line (T) to the fluorescence intensity of the quality control line (C) of each immunochromatographic time-resolved fluorescence test strip is obtained. The relationship curve between the ratio of the fluorescence intensity of the test line (T) to the fluorescence intensity of the quality control line (C) of the immunochromatographic time-resolved fluorescence test strip and the polystyrene concentration is obtained by fitting.

[0075] Finally, it should be noted that the above description is only a preferred embodiment of the present application and is not intended to limit the present application. Although the present application 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 make equivalent replacements to some technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A polystyrene micro / nanoplastics immunochromatographic time-resolved fluorescence reagent kit, characterized in that, The present invention comprises a fluorescent test strip and a sample reaction vial containing a lyophilized anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres. The fluorescent test strip includes a base plate, on which an absorbent pad, a detection pad, and a sample pad are sequentially attached from top to bottom on the adhesive side. Adjacent pads overlap at their joints. The detection pad is based on a nitrocellulose membrane, on which a horizontal control line and a detection line are arranged from top to bottom. The control line is coated with an 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, classified and named Hybridoma cell line PS-17, with the accession number CCTCC. NO:C2024145, deposited on May 10, 2024, deposited at the China Center for Type Culture Collection, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. The polystyrene micro / nanoplastics immunochromatographic time-resolved fluorescence reagent kit according to claim 1, characterized in that, The quantum dot fluorescent microsphere-labeled anti-polystyrene monoclonal antibody lyophilized product was prepared using the following steps: S11. The anti-polystyrene monoclonal antibody and the activated quantum dot fluorescent microsphere labeling reagent are mixed in borate buffer, shaken and reacted, and then centrifuged, reconstituted and blocked to obtain the quantum dot fluorescent microsphere labeled anti-polystyrene monoclonal antibody; wherein 1 mL of the activated quantum dot fluorescent microsphere labeling reagent is coupled with 40 pg to 90 ug of anti-polystyrene monoclonal antibody. S12. The quantum dot fluorescent microsphere-labeled anti-polystyrene monoclonal antibody prepared in step S11 is reconstituted in 0.01 mol / L pH 7.4 phosphate buffer containing 1.5% m / v trehalose and 2% m / v bovine serum albumin, and then lyophilized in a freeze dryer to obtain the lyophilized product of quantum dot fluorescent microsphere-labeled anti-polystyrene monoclonal antibody.

3. The polystyrene micro / nanoplastics immunochromatographic time-resolved fluorescence reagent kit according to claim 2, characterized in that, The activation method of the quantum dot fluorescent microsphere labeling reagent in step S11 includes the following steps: take the quantum dot microsphere labeling reagent, disperse it ultrasonically with pH 8.2 0.2M borate buffer, then slowly add carbodiimide solution, activate by shaking at room temperature, centrifuge to remove the supernatant, and reconstitute with pH 8.2 0.2M borate buffer for later use. The activation time is 15~30min.

4. The polystyrene micro / nanoplastics immunochromatographic time-resolved fluorescence reagent kit according to claim 1, characterized in that, The sample reaction bottle is a 1-5 mL bayonet bottle, and the content of the lyophilized anti-polystyrene monoclonal antibody labeled with quantum dot fluorescent microspheres in the sample reaction bottle is 100-200 ng.

5. The polystyrene micro / nanoplastics immunochromatographic time-resolved fluorescence reagent kit according to claim 1, characterized in that, The fluorescent test strip is prepared using the following steps: S21. Cut absorbent paper into absorbent pads; S22. Preparation of the detection pad: The polystyrene-hemocyanin conjugate is prepared into a coating solution with a concentration of 0.2~0.5 mg / mL using coating buffer. The conjugate is then applied laterally to the nitrocellulose membrane at a distance of 15~20 mm from the top edge using a line spraying method to obtain the detection line. The membrane is then dried at 37~40℃ for 60~120 minutes. The amount of polystyrene-hemocyanin conjugate required per centimeter of detection line is 80~400 ng. Prepare a coating solution of 0.2–0.5 mg / mL using coating buffer. Apply the coating solution laterally to a nitrocellulose membrane at a distance of 5–10 mm from the test line using a line spraying method to obtain a control line. Then, dry the membrane at 37–40 °C for 60–120 minutes. The amount of anti-mouse polyclonal antibody required per centimeter of control line is 100–300 ng. S23. Preparation of sample pad: Immerse the glass fiber membrane in the sealing solution, remove it, and dry it at 37~40℃ for 10~16 hours to obtain the sample pad. Then store it in a desiccator at room temperature. S24. Assembly of fluorescent test strips: On one side of the cardboard, from top to bottom, attach the absorbent pad, the detection pad, and the sample pad in sequence. The adjacent pads are overlapped at the joints, with an overlap length of 1~3mm, to obtain the fluorescent test strip.

6. The polystyrene micro / nanoplastics immunochromatographic time-resolved fluorescence reagent kit according to claim 5, characterized in that, The absorbent pad of 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 is 15-20 mm; and the distance between the control line and the detection line is 5-10 mm.

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

8. The polystyrene micro / nanoplastics immunochromatographic time-resolved fluorescence reagent kit according to claim 5, characterized in that, The blocking solution in step S23 is obtained by adding 2g ovalbumin, 2g sucrose, 0.02g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate dodecahydrate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate, and 0.5g Tween-20 to water to a final volume of 100mL.

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

10. An application of the polystyrene micro / nanoplastics immunochromatographic time-resolved fluorescence kit as described in any one of claims 1-9 for non-diagnostic purposes, wherein the kit is used to detect the content of polystyrene micro / nanoplastics.

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