A non-diagnostic purpose micro-nano plastic NC membrane one-step precipitation colorimetric detection method

Abstract

The application discloses a non-diagnostic purpose NC film one-step precipitation color development detection method for micro-nano plastics, comprising the following steps: preparing micro-nano plastic-OVA conjugate; preparing NC film coated with micro-nano plastic-OVA conjugate; placing the NC film into a polystyrene antibody solution and a sample solution, incubating at 37 DEG C for 1h, then washing once with PBST, and taking out; diluting goat anti-mouse lgG-HRP with 20% NBS-PBS in a centrifugal tube, and placing the NC film in the centrifugal tube, incubating at 37 DEG C for 30 min; washing the NC film 4-5 times, and absorbing water on a paper towel; after taking out the NC film after being soaked in precipitated TMB for 30s, absorbing water on a paper towel, and placing at room temperature after color development, the concentration of micro-nano plastics is detected. The application directly detects in a solution, does not need a sample pad and a water absorption pad, saves cost, does not need freeze-dried powder, and does not need a running strip, and realizes visual, qualitative and quantitative detection.

Description

TECHNICAL FIELD

[0001] The application relates to a non-diagnostic NC membrane one-step precipitation color development detection method for micro-nano plastics. BACKGROUND

[0002] Microplastics are a new type of environmental pollutants, 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, the number of microplastic particles is counted and all the microplastics are picked out and weighed, which is not only time-consuming and laborious, but also prone to errors in operation. The microscopic infrared spectroscopy and the microscopic Raman spectroscopy are basically the same as the visual method in the 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 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 non-diagnostic NC membrane one-step precipitation color development detection method for micro-nano plastics is urgently needed. SUMMARY

[0005] In order to solve the defects in the prior art, the application provides a non-diagnostic NC membrane one-step precipitation color development detection method for micro-nano plastics.

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

[0007] The application provides a non-diagnostic NC membrane one-step precipitation color development detection method for micro-nano plastics, which comprises the following steps:

[0008] S1, coupling micro-nano plastics with OVA to prepare micro-nano plastic-OVA conjugates;

[0009] S2, drawing lines on the NC membrane with micro-nano plastic-OVA to prepare an NC membrane coated with micro-nano plastic-OVA conjugates;

[0010] S3, put the NC membrane coated with micro-nano plastic-OVA conjugate into the polystyrene antibody solution and sample solution, incubate at 37°C for 1h, then wash the NC membrane with PBST once, and take it out; the polystyrene antibody is secreted by hybridoma cell line PS-8, the accession number of the hybridoma cell line PS-8 is CCTCC NO: C2024144, the preservation date is May 10, 2024, and the preservation unit is China Center for Type Culture Collection, Wuhan University, Wuhan, Hubei Province, China;

[0011] S4, dilute the goat anti-mouse lgG-HRP with 20% NBS-PBS in a centrifuge tube, and put the NC membrane in the centrifuge tube, incubate at 37°C for 30min;

[0012] S5, wash the NC membrane 4-5 times, and dry it on a paper towel, immerse the NC membrane in the precipitated TMB for 30s, then take it out, dry it on a paper towel, and place it at room temperature for color development, then detect the concentration of micro-nano plastic.

[0013] Preferably, the coupling of micro-nano plastic and OVA in step S1 includes the following steps:

[0014] S11, cool the microplastic, coupling buffer solution, and coupling washing / storage solution to room temperature;

[0015] S12, take the microplastic to a centrifuge tube, centrifuge, and then aspirate the supernatant, add coupling buffer solution for resuspension, centrifuge again, and then aspirate the supernatant, and resuspend with coupling buffer solution;

[0016] S13, dissolve the coupling carbodiimide (Polylink EDAC) in the coupling buffer solution to prepare an EDAC solution;

[0017] S14, add the EDAC solution to the microplastic suspension and mix well for activation;

[0018] S15, add OVA protein, dilute with coupling buffer solution to 1-5mg / mL, and mix well at room temperature for 30-60min;

[0019] S16, centrifuge and aspirate the supernatant;

[0020] S17, add 0.4mL coupling buffer solution for resuspension;

[0021] S18, repeat steps S16 and S17 to finally obtain micro-nano plastic-OVA conjugate, which is stored at 4°C.

[0022] Preferably, the detection of the concentration of micro-nano plastic in step S5 includes the following steps:

[0023] S51, take a photo of the NC film, and obtain the color depth of the precipitated TMB color development;

[0024] S52, convert the color depth of the precipitated TMB color development into a gray value, and construct a standard curve with the concentration;

[0025] S53, input the test gray value of the sample to be tested into the standard curve to obtain the concentration of micro-nano plastic in the sample to be tested.

[0026] Compared with the prior art, the present application has the following beneficial effects:

[0027] The present application directly detects in a solution (wet detection), without a sample pad and a water absorption pad, thereby saving costs, and at the same time, the present application does not need a freeze-dried powder and a running strip, and realizes visual, qualitative and quantitative detection.

[0028] Cell preservation:

[0029] The polystyrene antibody provided by the present application is secreted by a hybridoma cell line PS-8, the preservation number of the hybridoma cell line PS-8 is CCTCC NO: C2024144, the preservation date is May 10, 2024, and 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 City, Hubei Province. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 is a structural schematic diagram of a one-step precipitated color test paper for microplastic NC film for non-diagnostic purposes in the present application, the label (1) represents an NC film, the label (2) represents a p16-25 control antibody solution control line, and the label (3) represents PS-OVA;

[0031] Figure 2 is a detection result judgment standard for micro-nano plastic using an NC film for non-diagnostic purposes in the present application;

[0032] Figure 3 is a one-step precipitated color detection result for micro-nano plastic using an NC film for non-diagnostic purposes in the present application. DETAILED DESCRIPTION

[0033] The preferred embodiments of the present application are described below in combination with the drawings, and 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.

[0034] Example 1: coupling of microplastic (PS-CHOOH) and OVA.

[0035] 1. Cool microplastics, polylink coupling buffer and polylink wash / storage buffer to room temperature.

[0036] 2. Take 12.5 mg microplastics to 1.5 ml centrifuge tube, centrifuge at 500-1000 x g for 5 min, carefully aspirate supernatant, resuspend with 0.4 ml polylink coupling buffer, centrifuge at 500-1000 x g for 5 min, carefully aspirate supernatant, resuspend with 0.17 ml polylink coupling buffer.

[0037] 3. Dissolve 10 mg polylink EDAC in 50 uL ploylink coupling buffer to prepare 200 mg / ml EDAC solution, prepare fresh before use.

[0038] 4. Add 20 ul EDAC solution to the microplastics suspension, mix well for 15 min for activation.

[0039] 5. Add 500 ug OVA protein, diluted with ploylink coupling buffer to 5 mg / mL, rotate to mix well at room temperature for 30 min.

[0040] 6. Centrifuge at 500-1000 x g for 10 min, aspirate supernatant.

[0041] 7. Resuspend with 0.4 ml polylink wash / storage buffer, repeat steps 6 and 7 to obtain PS-OVA, finally store at 4°C.

[0042] 8. Perform SDS-PAGE analysis (12% gel).

[0043] Example 2: PS-OVA coated NC membrane.

[0044] 1. Take 30 uL PS-OVA and draw lines on the NC membrane with a line marker at a spraying amount of 0.08 ul / cm, take 30 uL 1 mg / mL p16 protein as a control and draw lines on another NC membrane, the NC membrane is as shown in Figure 1 ; p16 protein is a cell cycle protein-dependent kinase inhibitor, used as a positive control and a negative control.

[0045] 2. Place the NC membrane in a humidity below 15%, dry at 50°C overnight.

[0046] Example 3: NC membrane detection.

[0047] 1, PS-8 monoclonal antibody was diluted with 20% NBS-PBS to 2 μg / mL, and 2 mL of the antibody solution was taken in a 5 mL centrifuge tube; control monoclonal antibody p16-25 was diluted with 20% NBS-PBS to 2 μg / mL, and 2 mL of the antibody solution was taken in a 5 mL centrifuge tube.

[0048] 2, the PS-OVA coated NC film was cut into small pieces of the same width and placed in the PS antibody solution, p16-25 control antibody solution (as a negative control); the p16 coated NC film was also cut into small pieces of the same width and placed in the p16-25 control antibody solution, and another small piece was cut and placed in the PS-1 antibody solution as a negative control; the PS sample extraction diluent was added to the antibody solution.

[0049] 3, after incubation at 37°C for 1 h, the NC film was washed with PBST once.

[0050] 4, the goat anti-mouse lgG-HRP was diluted 1 W times with 20% NBS-PBS, and 10 mL was taken in a centrifuge tube, and the NC film was also placed in the centrifuge tube, and incubated at 37°C for 30 min.

[0051] 5, the NC film was washed 4-5 times, and the water was absorbed on a paper towel, the NC film was completely immersed in the precipitated TMB for 30 s, then taken out, the water was absorbed on a paper towel, and the color was developed at room temperature, and the results were photographed by a mobile intelligent detection device.

[0052] 6, the intelligent detection APP constructed a standard curve according to the color depth (converted into gray value) and concentration of the precipitated TMB, and the concentration of the micro-nano plastic in the sample was calculated by inputting the test gray value of the sample into the standard curve. As shown in Figure 2 , the results from left to right are negative, positive, and invalid, and the results measured in this example are shown in Figure 3 .

[0053] Example 4: Preparation of PS-8 monoclonal antibody.

[0054] (1) Microplastics (PS-CHOOH) coupling with conjugated proteins (BSA / KLH): Microplastics, coupling buffer solution (Polylink coupling buffer), coupling carbodiimide (Polylink EDAC), coupling buffer solution cooled to room temperature. Take 12.5 mg of microplastics into a 1.5 mL centrifuge tube, centrifuge at 500-1000 g for 10 min, discard the supernatant; add 0.4 mL of coupling buffer solution to resuspend, centrifuge again, 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 immediately before use. Add 20 μL of EDAC solution to the 0.17 mL of coupling buffer solution suspension, and fix it on a rotator for 15 min at room temperature for activation. Add 200 μg of conjugated protein (BSA / KLH), and fix it on a rotator for 60 min at room temperature for mixing. (Dissolve the protein in coupling buffer solution to 1 mg / ml). Centrifuge at 500-1000 g for 10 min, and aspirate the supernatant to detect the amount of conjugated protein (BSA / KLH). Add 0.4 mL of coupling wash / storage buffer (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 shown in FIG. 1, which proves that the coupling of PS-KLH and PS-BSA is successful. Figure 1

[0055] (2) Preparation of monoclonal antibodies: 6-8 week old female Balb / c mice are selected, and the prepared PS-BSA artificial antigen is used for immunization injection. Adult Balb / c mice receive primary immunization by subcutaneous administration (in the area between the shoulders), and receive booster immunization once 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 the cDNA, and are recombined on phages to construct a nanobody gene library. Then specific antibody screening is performed to obtain the required antibodies, the PS-BSA artificial antigen is fixed on a carrier, and then interacts with the phage library. Then the carrier surface is washed to wash away unbound or non-specifically bound phages, and strong acid is used to destroy the binding, thereby obtaining a positive phage solution. After 2-3 rounds of repetition, the required monoclonal antibodies are obtained.

[0056] ​The specific response of the immune system of Balb / c mice to the injection of PS-BSA artificial antigen was observed, and the mechanism of specific immune response of the antibody was explored.

[0057] Table 1: Immunization scheme of mice.

[0058]

[0059] The 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;

[0060] The amino acid sequence of CDR1 of the heavy chain variable region is shown in SEQ ID No. 1, 50-54;

[0061] The amino acid sequence of CDR2 of the heavy chain variable region is shown in SEQ ID No. 1, 69-85;

[0062] The amino acid sequence of CDR3 of the heavy chain variable region is shown in SEQ ID No. 1, 118-127;

[0063] The amino acid sequence of CDR1 of the light chain variable region is shown in SEQ ID No. 2, 44-54;

[0064] The amino acid sequence of CDR2 of the light chain variable region is shown in SEQ ID No. 2, 70-76;

[0065] The amino acid sequence of CDR3 of the light chain variable region is shown in SEQ ID No. 2, 109-117.

[0066] The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID No. 1:

[0067] MEWSWIFLFLLSGTAGVHSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCASSYYYGSSYGYWGQGTTLTVSS.

[0068] The amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID No. 2:

[0069] MESQIQVFVFVFLWLSGVDGDIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPLTFGAGTKLELK.

[0070] Finally, it should be noted that the above only for the preferred embodiments of the present application, and is not intended to limit the present application, although the foregoing detailed description of the present application has been made with reference to the foregoing embodiments, for those skilled in the art, it still can be modified, or part of the technical features of the equivalent replacement of the technical solutions described in the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application, should be included within the scope of the present application.

Claims

1. A one-step precipitation colorimetric detection method for micro- and nano-plastics for non-diagnostic purposes using NC membrane, characterized by, The method comprises the following steps: S1, coupling micro-nano plastic with OVA to prepare micro-nano plastic-OVA conjugate; S2, drawing a line on the NC membrane with the micro-nano plastic-OVA to prepare the NC membrane coated with the micro-nano plastic-OVA conjugate; S3, placing the NC membrane coated with the micro-nano plastic-OVA conjugate into a polystyrene antibody solution and a sample solution, incubating at 37 DEG C for 1 h, then washing the NC membrane with PBST once, and taking out; the polystyrene antibody is secreted by a hybridoma cell line PS-8, the preservation number of the hybridoma cell line PS-8 (Hybridoma cell line PS-8) is CCTCC NO: C2024144, 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; S4, diluting goat anti-mouse IgG-HRP with 20% NBS-PBS in a centrifuge tube, and placing the NC membrane in the centrifuge tube, and incubating at 37 DEG C for 30 min; S5, washing the NC membrane 4-5 times, absorbing water on a paper towel, taking out the NC membrane after being completely immersed in precipitated TMB for 30 s, absorbing water on a paper towel, and detecting the concentration of micro-nano plastic after color development at room temperature.

2. The method according to claim 1, wherein the method is characterized by, The coupling of micro-nano plastic and OVA in the step S1 comprises the following steps: S11, cooling micro-plastic, coupling buffer solution and coupling cleaning / storage solution to room temperature; S12, taking micro-plastic into a centrifuge tube, absorbing supernatant after centrifugation, adding coupling buffer solution for resuspension, absorbing supernatant again after centrifugation, and resuspending with coupling buffer solution; S13, dissolving carbodiimide in the coupling buffer solution to prepare an EDAC solution; S14, adding the EDAC solution to the micro-plastic suspension, mixing, and activating; S15, adding OVA protein, diluting with coupling buffer solution to 1-5 mg / mL, and rotating and mixing at room temperature for 30-60 min; S16, absorbing supernatant after centrifugation; S17, adding 0.4 ml of coupling buffer solution for resuspension; S18, repeating steps S16 and S17 to finally obtain micro-nano plastic-OVA conjugate, which is stored at 4 DEG C.

3. The method according to claim 1, wherein the method is characterized by, The detection of the concentration of micro-nano plastic in the step S5 comprises the following steps: S51, taking a photo of the NC membrane to obtain the color depth of the precipitated TMB color development; S52, converting the color depth of the precipitated TMB color development into a gray value, and constructing a calibration curve with the concentration; S53, inputting the gray value of the sample to be tested into the calibration curve to calculate the concentration of micro-nano plastic in the sample to be tested.

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