CN118032732A - Method for detecting MPs by covalent chemical labeling method - Google Patents
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- CN118032732A CN118032732A CN202410432687.XA CN202410432687A CN118032732A CN 118032732 A CN118032732 A CN 118032732A CN 202410432687 A CN202410432687 A CN 202410432687A CN 118032732 A CN118032732 A CN 118032732A
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- 239000000126 substance Substances 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 24
- 238000002372 labelling Methods 0.000 title claims abstract description 21
- 239000000243 solution Substances 0.000 claims abstract description 41
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 claims abstract description 40
- 239000007983 Tris buffer Substances 0.000 claims abstract description 36
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims abstract description 36
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims abstract description 26
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 22
- 238000001514 detection method Methods 0.000 claims abstract description 21
- 229960003638 dopamine Drugs 0.000 claims abstract description 20
- 239000012224 working solution Substances 0.000 claims abstract description 16
- 239000010414 supernatant solution Substances 0.000 claims abstract description 12
- 238000006243 chemical reaction Methods 0.000 claims abstract description 11
- 238000004140 cleaning Methods 0.000 claims abstract description 9
- 230000010355 oscillation Effects 0.000 claims abstract description 8
- 239000011550 stock solution Substances 0.000 claims abstract description 8
- 238000005303 weighing Methods 0.000 claims abstract description 3
- 229920001896 polybutyrate Polymers 0.000 claims description 5
- 239000002245 particle Substances 0.000 claims description 4
- 230000035484 reaction time Effects 0.000 claims description 2
- 229920000426 Microplastic Polymers 0.000 abstract description 44
- 230000007547 defect Effects 0.000 abstract description 4
- 238000007447 staining method Methods 0.000 abstract description 4
- 239000003344 environmental pollutant Substances 0.000 abstract description 3
- 239000004952 Polyamide Substances 0.000 description 5
- 239000004698 Polyethylene Substances 0.000 description 5
- 239000000975 dye Substances 0.000 description 5
- 229920002647 polyamide Polymers 0.000 description 5
- 229920000573 polyethylene Polymers 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 4
- 229920000747 poly(lactic acid) Polymers 0.000 description 4
- -1 polyethylene Polymers 0.000 description 4
- 239000004626 polylactic acid Substances 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 239000004743 Polypropylene Substances 0.000 description 3
- 229920001155 polypropylene Polymers 0.000 description 3
- 239000004793 Polystyrene Substances 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 238000002795 fluorescence method Methods 0.000 description 2
- 238000001215 fluorescent labelling Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000000053 physical method Methods 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 229920001690 polydopamine Polymers 0.000 description 2
- 239000004800 polyvinyl chloride Substances 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
- 238000001069 Raman spectroscopy Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 229920000704 biodegradable plastic Polymers 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000008602 contraction Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000001506 fluorescence spectroscopy Methods 0.000 description 1
- 238000003234 fluorescent labeling method Methods 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- VOFUROIFQGPCGE-UHFFFAOYSA-N nile red Chemical compound C1=CC=C2C3=NC4=CC=C(N(CC)CC)C=C4OC3=CC(=O)C2=C1 VOFUROIFQGPCGE-UHFFFAOYSA-N 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 238000011202 physical detection method Methods 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 229920002961 polybutylene succinate Polymers 0.000 description 1
- 239000004631 polybutylene succinate Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 229920000915 polyvinyl chloride Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 229920001169 thermoplastic Polymers 0.000 description 1
- 239000004416 thermosoftening plastic Substances 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a method for detecting MPs by a covalent chemical labeling method, which belongs to the technical field of environmental pollutant detection and comprises the following steps: s1, preparing a Tris solution of 50 mM; s2, preparing a FITC fluorescent dye stock solution, and then preparing a FITC working solution by using the Tris solution in S1; s3, preparing a dopamine Tris solution with a certain concentration; s4, weighing an MPs sample and a dopamine Tris solution for oscillation reaction; s5, after the reaction is finished, centrifuging to remove supernatant solution and reserving PDA-MPs; s6, cleaning the PDA-MPs, adding FITC working solution into the PDA-MPs, and reacting at room temperature; s7, centrifuging to remove supernatant solution, cleaning, and observing. The invention relates to a method for detecting MPs by a covalent chemical labeling method, which overcomes the defects of the existing physical fluorescent staining method and utilizes chemical characteristics to finish the detection of microplastic; can be aimed at microplastic with different types, sizes and characteristics, and has good universality.
Description
Technical Field
The invention relates to the technical field of environmental pollutant detection, in particular to a method for detecting MPs by a covalent chemical labeling method.
Background
Microplastic (MPs) are typically insoluble plastic chips, particles or fibrous films, etc., with a wide range of particle size distributions as small as 1 nm up to 5 mm. The chemical components of MPs mainly include Polystyrene (PS), polyethylene (PE), polyvinyl chloride (PVC), polypropylene (PP), polyamide (PA), polybutylene succinate (PBS, biodegradable), polylactic acid (PLA, which is a food-grade biodegradable material PLA), polybutylene terephthalate-adipate (PBAT, thermoplastic biodegradable plastic), and the like. MPs are widely distributed in the world, large in quantity, strong in toxicity and are carriers of pollutants, and even biological ingestion can finally influence human health through food chains, so that the MPs draw attention of the scientific community and become research hotspots of researchers.
The detection method of MPs mainly comprises a physical detection method and a chemical detection method. For example, direct observation of the number of MPs by the naked eye or by microscopy is the most direct physical assay. Common chemical detection methods include fourier transform infrared spectroscopy, raman spectroscopy, fluorescence spectroscopy, and chromatographic mass spectrometry. Among these methods, the application in the detection of MPs has received a lot of attention because the fluorescence method enables the direct physical method and the accuracy of the chemical method.
The key point of detecting MPs by using a fluorescence method is to perform effective fluorescent pre-dyeing treatment on a sample to be detected. Most of the prior art uses the physical adsorption of dye by MPs; for example, the invention patent publication No. CN110006723B discloses a method for performing fluorescent staining based on the thermal expansion and contraction characteristics of MPs. The method comprises the steps of firstly heating MPs powder dispersed in water solution containing DMSO, adding dye solution to dye for about 30 minutes after the temperature reaches more than 50 ℃, enabling dye to enter the heated and expanded plastic, then rapidly cooling in ice water mixed solution to enable MPs to shrink under cooling, and locking the dye in the MPs; the invention patent with publication number CN115993273A discloses that MPs are dyed under dark conditions by using a complex fluorescent dye of nile red and 4', 6-diamidino-2-phenylindole; these patents are all physical methods used, and the effect and stability of the dyeing may be affected by other factors.
Based on the above problems, the present invention proposes a method for MPs detection by covalent chemical labeling.
Disclosure of Invention
The invention aims to provide a method for detecting MPs by a covalent chemical labeling method, which overcomes the defects of the existing physical fluorescent staining method and utilizes chemical characteristics to finish the detection of microplastic; can be aimed at microplastic with different types, sizes and characteristics, and has good universality.
To achieve the above object, the present invention provides a method for detecting MPs by covalent chemical labeling, comprising the steps of:
S1, preparing a Tris solution of 50 mM;
S2, preparing a FITC fluorescent dye stock solution, and then preparing a FITC working solution by using the Tris solution in S1;
s3, preparing a dopamine Tris solution with a certain concentration;
s4, weighing a certain amount of MPs sample and a dopamine Tris solution for oscillation reaction;
s5, after the reaction is finished, centrifuging to remove supernatant solution and reserving PDA-MPs;
s6, cleaning the PDA-MPs, adding FITC working solution into the PDA-MPs after cleaning, and reacting at room temperature;
S7, centrifuging to remove supernatant solution, cleaning, and observing by using an inverted full-electric fluorescence microscope.
Preferably, in the step S2, the concentration of the FITC fluorescent dye stock solution is 0.1g/L, and the concentration of the FITC working solution is 0.1-10 mg/L.
Preferably, in the S3, the concentration of the dopamine Tris solution is 0.05-0.5 g/L.
Preferably, in the step S4, the mass of the MPs sample is 0.01-20 mg, the particle size is 1 nm-5 mm, and the type is one or more of PS, PE, PVC, PP, PA, PBS, PLA, PBAT.
Preferably, in the S4, the amount of the dopamine Tris solution is 1.0 mL.
Preferably, in the step S4, the temperature of the oscillation reaction is 26-70 ℃ and the oscillation time is 5-120 min.
Preferably, in the step S6, the dosage of the FITC working solution is 1.0 mL, and the reaction time is 15-60 min.
Preferably, in both S6 and S7, the amount of Tris solution is 0.5-2.0 mL.
Therefore, the method for detecting the MPs by the covalent chemical labeling method overcomes the defects of the existing physical fluorescent staining method, and forms a stable Polydopamine (PDA) film based on self-polymerization reaction of Dopamine (DA) on the surface of any material, wherein active amino on the PDA can be combined with fluorescein-5-isothiocyanate (FITC) through a covalent bond, so that the detection of the MPs is realized; the invention has good universality against different types, sizes and characteristics of microplastic.
The technical scheme of the invention is further described in detail through the drawings and the embodiments.
Drawings
FIG. 1 is an MPs observation diagram of the present invention in example 1, wherein A is spherical MPs; b is long-strip MPs; c is irregular MPs;
FIG. 2 is a diagram showing the observation of MPs labeled with different sizes in example 1 of the present invention.
Detailed Description
The technical scheme of the invention is further described below through the attached drawings and the embodiments.
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention.
Example 1
Labeling PA MPs using the covalent chemical fluorescent labeling method of the present invention:
Using 50mM Tris solution and 1.0 g/L FITC fluorescent dye stock solution, preparing 1.0 mg/L FITC fluorescent dye working solution and 0.25 g/L DA Tris solution;
1mL, 0.25: 0.25 g/L DA Tris solution was taken and added with 10 PA MPs of mg weighed, and after vortexing, 60: 60 min were reacted at 40℃with shaking: after the reaction, the supernatant solution was removed by centrifugation to leave PDA-MPs, and the PDA-MPs were washed 2 times with 1.0 mL Tris solution; then, 1.0 mL FITC fluorescent dye working solution was added to the washed PDA-MPs to react at room temperature for 30. 30min, and then the supernatant solution was removed by centrifugation and washed 2 times with 1.0 mL Tris solution, and then observed by using a ZEISS inverted all-electric fluorescence microscope.
Fluorescent microscope detection conditions: fluorescence photography was performed using a zeiss Axio ob server 7 system comprising a bingo camera and the Excelitas X-cite series XI120-QX. The microscope was equipped with a 38HE green fluorescence Prot filter (excitation wavelength 450-490nm, emission wavelength 500-550nm, dichroic 495 nm). Microplastic samples of different sizes and different morphologies were placed on a clean microscope object slide covered with a cover slip and photographed using Bright and FITC channels, respectively, with observations shown in fig. 1-2.
Example 2
The covalent chemical fluorescence labeling method is used for labeling PBAT MPs:
Using 50mM Tris solution and 1.0 g/L FITC fluorescent dye stock solution, preparing 1.0 mg/L FITC fluorescent dye working solution and 0.25 g/L DA Tris solution;
taking 1 mL and 0.25 g/L DA Tris solution, adding 10 mg PBAT MPs into the DA Tris solution, and carrying out vortex dispersion and then reacting at 40 ℃ under the condition of oscillation for 60 min; after the reaction, the supernatant solution was centrifuged to retain PDA-MPs, and the PDA-MPs were washed 2 times with 1.0 mL Tris solution; then, 1.0 mL of FITC fluorescent dye working solution was added to the washed PDA-MPs to react at room temperature for 30: 30 min, and then the supernatant solution was removed by centrifugation and washed 2 times with 1.0 mL of Tris solution, and then observed by using a ZEISS inverted all-electric fluorescence microscope.
The fluorescence microscope detection conditions were the same as in example 1.
Example 3
Labeling PE MPs by using the covalent chemical fluorescence labeling method of the invention:
Using 50mM Tris solution and 1.0 g/L FITC fluorescent dye stock solution, preparing 1.0 mg/L FITC fluorescent dye working solution and 0.25 g/L DA Tris solution;
Taking 1mL and 0.25 g/L DA Tris solution, adding the weighed 10mg PE MPs into the DA Tris solution, and carrying out vortex dispersion and then reacting at 40 ℃ under the oscillating condition for 60 min; after the reaction, the supernatant solution was centrifuged to retain PDA-MPs, and the PDA-MPs were washed 2 times with 1.0 mL Tris solution; then, 1.0 mL of FITC fluorescent dye working solution was added to the washed PDA-MPs to react at room temperature for 30: 30min, and then the supernatant solution was removed by centrifugation and washed 2 times with 1.0 mL of Tris solution, and then observed by using a ZEISS inverted all-electric fluorescence microscope.
The fluorescence microscope detection conditions were the same as in example 1.
Therefore, the method for detecting the MPs by the covalent chemical labeling method overcomes the defects of the existing physical fluorescent staining method and utilizes chemical characteristics to finish the detection of the microplastic; can be aimed at microplastic with different types, sizes and characteristics, and has good universality.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention and not for limiting it, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that: the technical scheme of the invention can be modified or replaced by the same, and the modified technical scheme cannot deviate from the spirit and scope of the technical scheme of the invention.
Claims (8)
1. A method for the detection of MPs by covalent chemical labelling, comprising the steps of:
S1, preparing a Tris solution of 50 mM;
S2, preparing a FITC fluorescent dye stock solution, and then preparing a FITC working solution by using the Tris solution in S1;
s3, preparing a dopamine Tris solution with a certain concentration;
s4, weighing a certain amount of MPs sample and a dopamine Tris solution for oscillation reaction;
s5, after the reaction is finished, centrifuging to remove supernatant solution and reserving PDA-MPs;
s6, cleaning the PDA-MPs, adding FITC working solution into the PDA-MPs after cleaning, and reacting at room temperature;
S7, centrifuging to remove supernatant solution, cleaning, and observing by using an inverted full-electric fluorescence microscope.
2. A method for the detection of MPs by covalent chemical labeling according to claim 1, characterized in that: in the step S2, the concentration of the FITC fluorescent dye stock solution is 0.1g/L, and the concentration of the FITC working solution is 0.1-10 mg/L.
3. A method for the detection of MPs by covalent chemical labeling according to claim 2, characterized in that: in the S3, the concentration of the dopamine Tris solution is 0.05-0.5 g/L.
4. A method for the detection of MPs by covalent chemical labelling according to claim 3, characterized in that: in the S4, the mass of the MPs sample is 0.01-20 mg, the particle size is 1 nm-5 mm, and the type is one or more of PS, PE, PVC, PP, PA, PBS, PLA, PBAT.
5. The method for detecting MPs by a covalent chemical labeling method according to claim 4, wherein: in the S4, the usage amount of the dopamine Tris solution is 1.0 mL.
6. A method for the detection of MPs by covalent chemical labeling according to claim 5, characterized in that: in the step S4, the temperature of the oscillation reaction is 26-70 ℃, and the oscillation time is 5-120 min.
7. A method for the detection of MPs by covalent chemical labeling according to claim 6, characterized in that: in the step S6, the dosage of the FITC working solution is 1.0 mL, and the reaction time is 15-60 min.
8. A method for the detection of MPs by covalent chemical labeling according to claim 7, characterized in that: in the S6 and the S7, the Tris solution is adopted for cleaning for 1-3 times, and the dosage of the Tris solution is 0.5-2.0 mL.
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| WO2023194414A1 (en) * | 2022-04-04 | 2023-10-12 | Cambridge Enterprise Limited | Polydopamine co-polymer nanoparticles |
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| CN106525788A (en) * | 2016-10-31 | 2017-03-22 | 甘肃省科学院传感技术研究所 | Preparation method of bionic nanometer film and method for fixing probe with same |
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| CN107383871A (en) * | 2017-07-21 | 2017-11-24 | 安徽师范大学 | A kind of preparation and application of the poly-dopamine composite of the load glucose oxidase of fluorescein isothiocynate modification |
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