CN110006723B - Fluorescent dyeing method for quantifying micro plastic based on expansion with heat and contraction with cold characteristics - Google Patents
Fluorescent dyeing method for quantifying micro plastic based on expansion with heat and contraction with cold characteristics Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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Abstract
The invention discloses a fluorescent dyeing marking method of micro-plastic, which is characterized by comprising the following steps: and (3) carrying out fluorescent dyeing marking on the micro plastic by adopting a physical method based on the characteristics of expansion with heat and contraction with cold. The invention also provides a fluorescent staining marking method for quantifying the micro-plastics, which is characterized by comprising the following steps: the method comprises the following steps: preparing a dye solution, coloring the micro plastic powder, filtering, and calculating the recovery rate of the micro plastic powder. The invention utilizes the expansion and contraction characteristics of the plastic to make the plastic marked by the fluorescent dye, thereby leading the micro plastic to be more easily and rapidly identified, namely, based on the expansion and contraction characteristics of the plastic, the plastic is heated and expanded under the heating state, the dye is added at the moment to facilitate the plastic to enter the plastic, the plastic is rapidly cooled after being heated for a certain time, and the plastic is locked in the plastic due to the contraction and cooling, thereby leading the plastic to be marked by the fluorescent dye.
Description
Technical Field
The invention relates to a fluorescent dyeing method for quantifying micro plastic based on expansion with heat and contraction with cold characteristics.
Background
As an important pollutant in aquatic environments, microplastics (< 5 mm) are gaining increasing global attention. The micro plastic is easy to adsorb various organic pollutants, metal elements, even pathogenic bacteria and the like due to small particles and large specific surface area, and the pollutants adsorbed on the micro plastic and additives contained in the micro plastic can generate compound toxic effect on marine organisms, so that the ingestion efficiency of the marine organisms is reduced, and the marine organisms are injured and even die. Marine and offshore organisms mistaking these micro-plastics of similar density and size to those of low trophic level organisms can also cause the digestive tract of these marine organisms to clog, causing pseudo-satiety. The micro plastic adsorbing various pollutants has influence on the whole ecological system through the enrichment and the transmission of a food chain, and even finally influences the human body health. The world places more and more importance on the harm of the micro-plastic, the investigation and research of the micro-plastic in China are still in the initial stage, the public has low cognition degree on the current situation of the micro-plastic pollution and the harm thereof, and the public is lack of understanding on the ecological effect caused by the micro-plastic pollution.
It is reported that over 3 million tons of plastic are produced worldwide each year, with about 10% of the plastic going into the ocean. According to investigation, the weight of plastic garbage fragments discharged into the ocean every year in China is 132-353 million tons, china is considered as one of the largest plastic product producing and consuming countries, and the ocean ecosystem is seriously polluted by micro plastics. Waste plastics have various ways to flow into the ocean due to insufficient execution of waste management system and low recovery rate of plastic products. Fibers contained in water for washing clothes, and micro plastic particles contained in skin care products and detergents, which flow into rivers and oceans along with wastewater. The plastic garbage discarded by human beings on the beach at will, the use and the wear and aging of plastic fishing gear in the aquaculture, plastic particles remained on the beach or imported into the ocean in the processes of plastic product processing, maritime transportation and the like, and the plastic garbage is gradually broken or photodegraded through physical and chemical actions to be broken into micro plastics with different shapes. The diffusion range of the micro-plastics suspended on the sea surface is wider and wider along with the flow of the ocean currents, and the micro-plastics enter the coastal mudflat along with the ocean currents.
Micro-plastics constitute a significant environmental problem due to their threat to marine life and to humans. In order to further understand the influence of the micro-plastic on the environment, the identification of the micro-plastic is the most urgent problem to be solved. At present, the identification method of the micro-plastic in the environment generally uses a fluorescent dyeing method to assist identification, but the selection of the dye and the type of the dyed micro-plastic have certain limitations, and the dyeing method has the defects of complexity, long time consumption and the like. For example, the use of nile red staining allows rapid identification of microplastics based on the binding of dye molecules to the surface of the microplastics during contact with the surface, thereby allowing the microplastics to be fluorescently labeled, but this approach is complicated to pre-treat with nile red dye and has certain limitations on the type of microplastics stained (not applicable to polyvinyl chloride, polyamides and polyesters).
The invention utilizes the expansion and contraction characteristics of the plastic to make the plastic marked by the fluorescent dye, thereby enabling the micro plastic to be more easily and quickly identified and providing a reliable method for the further development of the research on the micro plastic.
Disclosure of Invention
The invention aims to provide a fluorescent staining and marking method of micro plastic, which is characterized by comprising the following steps: and (3) carrying out fluorescent dyeing marking on the micro plastic by adopting a physical method based on the characteristics of expansion with heat and contraction with cold.
The fluorescent staining marking method of the micro plastic is characterized by comprising the following steps: the method comprises the following steps: heating the micro plastic in a mixed solution of water and DMSO (the volume ratio is 1:9 to 9:1, and the micro plastic is miscible in any proportion) in a heating state to expand; and adding a dye solution into the mixed solution so as to facilitate the dye solution to enter the interior of the micro plastic, heating for a specified time, rapidly cooling in the ice-water mixed solution for 5min, and taking out to lock the dye in the interior of the plastic, so that the micro plastic is marked by the fluorescent dye.
The fluorescent staining marking method of the micro plastic is characterized by comprising the following steps: the micro plastic comprises at least one of four plastic powders of Polyethylene (PE), polystyrene (PS), polyvinyl chloride (PVC) and polyethylene terephthalate (PET).
The fluorescent staining marking method of the micro plastic is characterized in that: the heating temperature of the mixed solution is selected from any temperature range of 25-75 ℃.
The fluorescent staining marking method of the micro plastic is characterized in that: the dyeing time is selected from any time of 10 minutes to 30 minutes.
The fluorescent staining marking method of the micro plastic is characterized in that: the dye solution is selected from Nile Red (NR) dye, fluorescein Isothiocyanate (FITC) or safranin dye.
Another aspect of the present invention provides a fluorescent staining labeling method for quantifying a micro plastic, comprising: the method comprises the following steps: preparing a dye solution, coloring the micro plastic powder, filtering, and calculating the recovery rate of the micro plastic powder.
The fluorescent staining marking method of the quantitative micro plastic is characterized by comprising the following steps:
the dye solution is selected from a Nile Red (NR) dye dissolved in dimethyl sulfoxide (DMSO) to prepare a Nile red dye solution with the concentration of 500 mu g/mL, or a Fluorescein Isothiocyanate (FITC) and a safranin dye dissolved in pure water to prepare a dye solution with the concentration of 500 mu g/mL.
The fluorescent staining marking method of the quantitative micro plastic is characterized by comprising the following steps:
the coloring step of the micro plastic powder is that the micro plastic powder selected from Polyethylene (PE), polystyrene (PS), polyvinyl chloride (PVC) and polyethylene terephthalate (PET) is added into a mixed solution of water and DMSO with the volume ratio of 1:9 to 9:1, and the mixed solution is miscible in any proportion;
heating at room temperature to 75 ℃, adding the dye solution for dyeing when the temperature reaches 50 ℃ or 75 ℃, quickly cooling in the ice-water mixed solution for 5 minutes after dyeing for 10 to 30 minutes, and taking out.
The fluorescent staining marking method of the quantitative micro plastic is characterized by comprising the following steps: and the step of suction filtration is that the cooled sample liquid is suction filtered by a polytetrafluoroethylene filter membrane, washed by a small amount of DMSO solution to remove redundant dye solution, and washed by pure water until the solution is colorless.
The fluorescent staining marking method of the quantitative micro plastic is characterized by comprising the following steps: the step of calculating the recovery rate of the micro-plastic powder comprises the steps of placing the cell counting plate under a fluorescence inverted microscope for observation, and photographing and recording the micro-plastic conditions in a bright field and a fluorescence state by using a CCD (charge coupled device) so as to count and calculate the recovery rate of the micro-plastic powder.
The invention utilizes the expansion and contraction characteristics of the plastic to make the plastic marked by the fluorescent dye, thereby leading the micro plastic to be more easily and rapidly identified, namely, based on the expansion and contraction characteristics of the plastic, the plastic is heated and expanded under the heating state, the dye is added at the moment to facilitate the plastic to enter the plastic, the plastic is rapidly cooled after being heated for a certain time, and the plastic is locked in the plastic due to the contraction and cooling, thereby leading the plastic to be marked by the fluorescent dye. In addition, the method of the invention is applicable to a wide range of plastics. But because the chemical staining method is long in time and the required reagents are expensive, the method is simpler and quicker compared with a physical staining method based on expansion and contraction, the sample retention time is long, and the method is not limited to the selection of dyes and the type of micro-plastics, and provides a reliable method for identifying the micro-plastics in the environment. By the staining method of the invention, the existing micro-plastics in organisms and in the environment can be widely identified and quantified, and the method can be used for evaluating the potential risks of the micro-plastics in the environment.
Drawings
FIG. 1 is a bright field microscope and a fluorescence microscope after Nile Red staining;
FIG. 2 is the identification of four plastic powders under a bright field microscope and a fluorescent microscope after FITC staining;
FIG. 3 is an identification of four plastic powders under a bright field microscope and a fluorescent microscope after safranin staining;
FIG. 4 shows the identification of four plastic powders under bright field and fluorescent microscopes after FITC-silanization staining;
FIG. 5 is a comparison of the NR dyed four plastic powders before (left) and after (right) two months of retention in the physical dyeing process;
FIG. 6 is a comparison of four plastic powders dyed in the chemical dyeing process before (left) and after (right) two months;
FIG. 7 is a scanning electron micrograph of a standard (left) and heated at 75 deg.C (right) of four plastic powders;
FIG. 8 shows Raman spectra of four plastic powders before and after staining with Nile Red, FITC, and safranin, respectively, at 75 deg.C: a-PE; b-PVC; c-PS; d-PET.
Detailed Description
The technical solutions in the embodiments of the present invention will be described in detail below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
According to the invention, based on the expansion with heat and contraction with cold of the plastic, the dye is added when the plastic is heated and expanded in a heating state so as to be convenient for entering the plastic, the plastic is rapidly cooled after being heated for a certain time, and the dye is locked in the plastic due to the shrinkage with cold of the plastic, so that the plastic is marked by the fluorescent dye.
In the present invention, FIG. 1 is a bright field microscope and a fluorescence microscope after Nile Red staining; FIG. 2 is the identification of four plastic powders under a bright field microscope and a fluorescent microscope after FITC staining; FIG. 3 is an identification of four plastic powders under a bright field microscope and a fluorescent microscope after safranin staining; FIG. 4 shows the identification of four plastic powders under a bright field microscope and a fluorescence microscope after dyeing by FITC-silanization; FIG. 5 is a comparison of the NR dyed four plastic powders retained two months before (left) and after (right) in the physical dyeing process; FIG. 6 is a comparison of four plastic powders dyed in the chemical dyeing process before (left) and after (right) two months; FIG. 7 is a scanning electron micrograph of a standard (left) and a heating at 75 deg.C (right) of four plastic powders; FIG. 8 shows Raman spectra of four plastic powders before and after staining with Nile Red, FITC, and safranin, respectively, at 75 deg.C: a-PE; b-PVC; c-PS; d-PET.
Preparing different dye solution for standby:
weighing 0.05g of Nile Red (NR) dye, dissolving the nile red dye in 100mL of dimethyl sulfoxide (DMSO) to prepare a nile red dye solution with the concentration of 500 mu g/mL for later use; 0.05g of Fluorescein Isothiocyanate (FITC) and safranine dye are weighed and dissolved in 100mL of pure water to prepare a dye solution with the concentration of 500 mu g/mL, the FITC dye solution is wrapped by tinfoil and protected from light, and the dye solution is stored for standby at room temperature.
Coloring of the micro-plastic powder:
0.05g of four plastic powders of Polyethylene (PE), polystyrene (PS), polyvinyl chloride (PVC) and polyethylene terephthalate (PET) are weighed respectively, and then 49mL of mixed solution of water and DMSO (volume ratio of 1:9 to 9:1, miscible in any proportion) is added respectively.
Then placing the sample liquid in a room temperature, a temperature of 50 ℃ and a temperature of 75 ℃ for heating respectively, adding 1mL of nile red dye when the temperature reaches 50 ℃ or 75 ℃, starting to record dyeing time, taking out 10mL of sample liquid in a 15mL centrifuge tube when the dyeing time is 10min, then rapidly cooling in an ice-water mixed solution for 5min, then taking out the sample liquid, after dyeing for 20min and 30min, likewise respectively taking out 10mL of dyed sample liquid, rapidly placing in the ice-water mixed solution for cooling for 5min, and then taking out the sample liquid. And (3) carrying out suction filtration on the cooled sample liquid by using a polytetrafluoroethylene filter membrane, washing by using a small amount of DMSO (dimethylsulfoxide) solution to remove redundant Nile red dye, and washing by using pure water until the solution is colorless.
And finally, putting the filter membrane obtained after suction filtration into 20mL of ultrapure water for ultrasonic treatment to ultrasonically treat the micro-plastic powder on the membrane into the pure water, thereby obtaining a sample solution containing the dyed micro-plastic powder. The FITC and safranin dye staining procedure was the same as the nile red staining procedure.
And (3) taking 10 mu L of the sample solution, placing the sample solution on a cell counting plate, observing the sample solution under a fluorescence inverted microscope, and taking a CCD picture to record the micro-plastic conditions in a bright field and a fluorescence state so as to count and calculate the recovery rate of the micro-plastic powder.
In the invention, a physical dyeing method is adopted, namely, based on the expansion with heat and contraction with cold of the plastic, the dye is added when the plastic is heated and expanded in a heating state so as to be convenient for the plastic to enter the plastic, the plastic is rapidly cooled after being heated for a certain time, and the dye is locked in the plastic due to the contraction with cold of the plastic, so that the plastic is marked by the fluorescent dye.
In the invention, 0.05g of Nile Red (NR) dye is weighed and dissolved in 100mL of dimethyl sulfoxide (DMSO) to prepare a nile red dye solution with the concentration of 500 mu g/mL, 0.05g of Fluorescein Isothiocyanate (FITC) and safranine dye are weighed and dissolved in 100mL of pure water to prepare a dye solution with the concentration of 500 mu g/mL, and the FITC dye solution is wrapped by tinfoil and protected from light and is stored at room temperature for later use.
Examples
Example 1: process for dyeing polyethylene plastic powder with nile red based on thermal expansion and cold shrinkage method
(1) First, 0.05g of polyethylene plastic powder was weighed into a 250mL beaker, and 49mL of a mixed solution of water and DMSO (volume ratio of 1:9 to 9:1, miscible in any ratio) was added.
(2) The beaker was heated at room temperature, 50 ℃ and 75 ℃ respectively, and after the temperature reached, 1mL of Nile Red dye (500. Mu.g/mL) was added to start staining and the staining time was recorded.
(3) After the dyeing time reaches 10min, uniformly taking out 10mL of dyeing sample liquid from the beaker, quickly cooling the dyeing sample liquid in the ice-water mixed liquid, and after the dyeing time reaches 20min and 30min, respectively uniformly taking out 10mL of dyeing sample liquid, quickly placing the dyeing sample liquid in the ice-water mixed liquid, and cooling the dyeing sample liquid. Cooling for 5min and taking out.
(4) And (3) carrying out suction filtration on the cooled dyeing sample liquid by using a polytetrafluoroethylene filter membrane, washing by using a small amount of DMSO (dimethylsulfoxide) solution to remove redundant nile red dye, and washing by using pure water until the solution is colorless.
(5) And finally, putting the filter membrane obtained after suction filtration into 20mL of ultrapure water for ultrasonic treatment to ultrasonically treat the polyethylene plastic powder on the membrane into the pure water, thereby obtaining a sample solution containing the dyed polyethylene plastic powder.
(6) And (3) taking 10 mu L of the sample liquid on a cell counting plate, placing the sample liquid under a fluorescence inverted microscope for observation, and taking a picture by using a CCD (charge coupled device) to record the polyethylene plastic dyeing condition in a bright field and a fluorescence state so as to count and calculate the recovery rate of the micro plastic powder.
Example 2: process for dyeing polyvinyl chloride plastic powder with nile red based on thermal expansion and cold shrinkage method
(1) 0.05g of polyvinyl chloride plastic powder is first weighed into a 250mL beaker, and 49mL of a mixed solution of water and DMSO (volume ratio of 1:9 to 9:1, miscible in any ratio) is added.
(2) The beaker was heated at room temperature, 50 ℃ and 75 ℃ respectively, and after the temperature reached, 1mL of Nile Red dye (500. Mu.g/mL) was added to start staining and the staining time was recorded.
(3) After the dyeing time reaches 10min, uniformly taking out 10mL of dyeing sample liquid from the beaker, quickly cooling the dyeing sample liquid in the ice-water mixed liquid, and after the dyeing time reaches 20min and 30min, respectively uniformly taking out 10mL of dyeing sample liquid, quickly placing the dyeing sample liquid in the ice-water mixed liquid, and cooling the dyeing sample liquid. Cooling for 5min and taking out.
(4) And (3) carrying out suction filtration on the cooled dyeing sample liquid by using a polytetrafluoroethylene filter membrane, washing by using a small amount of DMSO (dimethyl sulfoxide) solution to remove redundant Nile red dye, and washing by using pure water until the solution is colorless.
(5) And finally, putting the filter membrane obtained after suction filtration into 20mL of ultrapure water for ultrasonic treatment to ultrasonically treat the polyvinyl chloride plastic powder on the membrane into the pure water, thereby obtaining a sample solution containing the dyed polyvinyl chloride plastic powder.
(6) And (3) taking 10 mu L of the sample liquid on a cell counting plate, placing the sample liquid under a fluorescence inverted microscope for observation, and taking a picture by using a CCD (charge coupled device) to record the polyvinyl chloride plastic dyeing conditions in a bright field and a fluorescence state so as to count and calculate the recovery rate of the micro plastic powder.
Example 3: process for dyeing polystyrene plastic powder with nile red based on thermal expansion and cold shrinkage method
(1) 0.05g of polystyrene plastic powder is initially weighed into a 250mL beaker, and 49mL of a mixed solution of water and DMSO (volume ratio 1:9 to 9:1, miscible in any ratio) are added.
(2) The beaker was heated at room temperature, 50 ℃ and 75 ℃ respectively, and after the temperature reached, 1mL of Nile Red dye (500. Mu.g/mL) was added to start staining and the staining time was recorded.
(3) After the dyeing time reaches 10min, uniformly taking 10mL of dyeing sample liquid out of the beaker and quickly cooling the dyeing sample liquid in the ice-water mixed liquid, and after the dyeing time reaches 20min and 30min, respectively uniformly taking 10mL of dyeing sample liquid out and quickly cooling the dyeing sample liquid in the ice-water mixed liquid. Cooling for 5min and taking out.
(4) And (3) carrying out suction filtration on the cooled dyeing sample liquid by using a polytetrafluoroethylene filter membrane, washing by using a small amount of DMSO (dimethylsulfoxide) solution to remove redundant nile red dye, and washing by using pure water until the solution is colorless.
(5) And finally, putting the filter membrane obtained after suction filtration into 20mL of ultrapure water for ultrasonic treatment to ultrasonically treat the polystyrene plastic powder on the membrane into the pure water, thereby obtaining a sample solution containing the dyed polystyrene plastic powder.
(6) And (3) taking 10 mu L of the sample liquid on a cell counting plate, placing the sample liquid under a fluorescence inverted microscope for observation, and taking a picture by using a CCD (charge coupled device) to record the staining condition of the polystyrene plastic in a bright field and a fluorescence state so as to count and calculate the recovery rate of the micro-plastic powder.
Example 4: process for dyeing polyethylene terephthalate plastic powder with nile red based on thermal expansion and cold shrinkage
(1) First, 0.05g of polyethylene terephthalate plastic powder was weighed into a 250mL beaker, and 49mL of a mixed solution of water and DMSO (1:9 to 9:1 by volume, miscible in any ratio) was added.
(2) The beaker was heated at room temperature, 50 ℃ and 75 ℃ respectively, and after the temperature reached, 1mL of Nile Red dye (500. Mu.g/mL) was added to start staining and the staining time was recorded.
(3) After the dyeing time reaches 10min, uniformly taking out 10mL of dyeing sample liquid from the beaker, quickly cooling the dyeing sample liquid in the ice-water mixed liquid, and after the dyeing time reaches 20min and 30min, respectively uniformly taking out 10mL of dyeing sample liquid, quickly placing the dyeing sample liquid in the ice-water mixed liquid, and cooling the dyeing sample liquid. Cooling for 5min and taking out.
(4) And (3) carrying out suction filtration on the cooled dyeing sample liquid by using a polytetrafluoroethylene filter membrane, washing by using a small amount of DMSO (dimethyl sulfoxide) solution to remove redundant Nile red dye, and washing by using pure water until the solution is colorless.
(5) And finally, putting the filter membrane obtained after suction filtration into 20mL of ultrapure water for ultrasonic treatment to ultrasonically treat the polyethylene terephthalate plastic powder on the membrane into the pure water, thereby obtaining a sample solution containing the dyed polyethylene terephthalate plastic powder.
(6) And (3) taking 10 mu L of the sample liquid on a cell counting plate, placing the sample liquid under a fluorescence inverted microscope for observation, and taking a picture by using a CCD (charge coupled device) to record the dyeing condition of the polyethylene terephthalate plastics in a bright field and a fluorescence state so as to count and calculate the recovery rate of the micro-plastic powder.
Table 1 shows the recovery rate of the nile red dye for four plastic powders, PE, PS, PVC and PET at room temperature, 50 ℃, 75 ℃ and dyeing time of 10min, 20min and 30 min.
Example 5: process for dyeing polyethylene plastic powder with FITC based on thermal expansion and cold shrinkage method
(1) First, 0.05g of polyethylene plastic powder was weighed into a 250mL beaker, and 49mL of a mixed solution of water and DMSO (volume ratio of 1:9 to 9:1, miscible in any ratio) was added.
(2) The beaker was left to warm at room temperature, 50 ℃ and 75 ℃ respectively, and after the temperature had reached, 1mL of FITC dye (500. Mu.g/mL) was added, staining was started and the staining time was recorded.
(3) After the dyeing time reaches 10min, uniformly taking out 10mL of dyeing sample liquid from the beaker, quickly cooling the dyeing sample liquid in the ice-water mixed liquid, and after the dyeing time reaches 20min and 30min, respectively uniformly taking out 10mL of dyeing sample liquid, quickly placing the dyeing sample liquid in the ice-water mixed liquid, and cooling the dyeing sample liquid. Cooling for 5min and taking out.
(4) The cooled dye sample solution was filtered with a teflon filter and washed with a large amount of pure water to wash the dye in the solution.
(5) And finally, putting the filter membrane obtained after suction filtration into 20mL of ultrapure water for ultrasonic treatment to ultrasonically treat the polyethylene plastic powder on the membrane into the pure water, thereby obtaining a sample solution containing the dyed polyethylene plastic powder.
(6) And (3) taking 10 mu L of the sample liquid on a cell counting plate, placing the sample liquid under a fluorescence inverted microscope for observation, and taking a picture by using a CCD (charge coupled device) to record the polyethylene plastic dyeing condition in a bright field and a fluorescence state so as to count and calculate the recovery rate of the micro plastic powder.
Example 6: procedure (1) for dyeing polyvinyl chloride plastic powder with FITC based on the thermal expansion and contraction method 0.05g of polyvinyl chloride plastic powder was first weighed into a 250mL beaker, and 49mL of a mixed solution of water and DMSO (volume ratio 1:9 to 9:1, miscible in any ratio) was added.
(2) The beaker was heated at room temperature, 50 ℃ and 75 ℃ respectively, and after the temperature reached, 1mL of FITC dye (500. Mu.g/mL) was added to start staining and the staining time was recorded.
(3) After the dyeing time reaches 10min, uniformly taking out 10mL of dyeing sample liquid from the beaker, quickly cooling the dyeing sample liquid in the ice-water mixed liquid, and after the dyeing time reaches 20min and 30min, respectively uniformly taking out 10mL of dyeing sample liquid, quickly placing the dyeing sample liquid in the ice-water mixed liquid, and cooling the dyeing sample liquid. Cooling for 5min and taking out.
(4) The cooled dye sample solution was filtered with a teflon filter and washed with a large amount of pure water to wash the dye in the solution.
(5) And finally, putting the filter membrane obtained after suction filtration into 20mL of ultrapure water for ultrasonic treatment to ultrasonically treat the polyvinyl chloride plastic powder on the membrane into the pure water, thereby obtaining a sample solution containing the dyed polyvinyl chloride plastic powder.
(6) And (3) taking 10 mu L of the sample liquid on a cell counting plate, placing the sample liquid under a fluorescence inverted microscope for observation, and taking a picture by using a CCD (charge coupled device) to record the polyvinyl chloride plastic dyeing conditions in a bright field and a fluorescence state so as to count and calculate the recovery rate of the micro plastic powder.
Example 7: process for dyeing polystyrene plastic powder with FITC based on thermal expansion and cold shrinkage method
(1) 0.05g of polystyrene plastic powder is initially weighed into a 250mL beaker, 49mL of a mixed solution of water and DMSO (1: 1) are added.
(2) The beaker was heated at room temperature, 50 ℃ and 75 ℃ respectively, and after the temperature reached, 1mL of FITC dye (500. Mu.g/mL) was added to start staining and the staining time was recorded.
(3) After the dyeing time reaches 10min, uniformly taking out 10mL of dyeing sample liquid from the beaker, quickly cooling the dyeing sample liquid in the ice-water mixed liquid, and after the dyeing time reaches 20min and 30min, respectively uniformly taking out 10mL of dyeing sample liquid, quickly placing the dyeing sample liquid in the ice-water mixed liquid, and cooling the dyeing sample liquid. Cooling for 5min and taking out.
(4) The cooled dye sample solution was filtered with a teflon filter and washed with a large amount of pure water to wash the dye in the solution.
(5) And finally, putting the filter membrane obtained after suction filtration into 20mL of ultrapure water for ultrasonic treatment so as to ultrasonically treat the polystyrene plastic powder on the membrane into the pure water, thereby obtaining a sample solution containing the dyed polystyrene plastic powder.
(6) And (3) taking 10 mu L of the sample liquid on a cell counting plate, placing the sample liquid under a fluorescence inverted microscope for observation, and taking a picture by using a CCD (charge coupled device) to record the staining condition of the polystyrene plastic in a bright field and a fluorescence state so as to count and calculate the recovery rate of the micro-plastic powder.
Example 8: process for dyeing polyethylene terephthalate plastic powder with FITC based on thermal expansion and cold shrinkage method
(1) 0.05g of polyethylene terephthalate plastic powder is first weighed into a 250mL beaker, and 49mL of a mixed solution of water and DMSO (volume ratio 1:9 to 9:1, miscible in any ratio) are added.
(2) The beaker was heated at room temperature, 50 ℃ and 75 ℃ respectively, and after the temperature reached, 1mL of FITC dye (500. Mu.g/mL) was added to start staining and the staining time was recorded.
(3) After the dyeing time reaches 10min, uniformly taking out 10mL of dyeing sample liquid from the beaker, quickly cooling the dyeing sample liquid in the ice-water mixed liquid, and after the dyeing time reaches 20min and 30min, respectively uniformly taking out 10mL of dyeing sample liquid, quickly placing the dyeing sample liquid in the ice-water mixed liquid, and cooling the dyeing sample liquid. Cooling for 5min and taking out.
(4) The cooled dye sample solution was filtered with a teflon filter and washed with a large amount of pure water to wash the dye in the solution.
(5) And finally, putting the filter membrane obtained after suction filtration into 20mL of ultrapure water for ultrasonic treatment to ultrasonically treat the polyethylene terephthalate plastic powder on the membrane into the pure water, thereby obtaining a sample solution containing the dyed polyethylene terephthalate plastic powder.
(6) And (3) taking 10 mu L of the sample liquid on a cell counting plate, placing the sample liquid under a fluorescence inverted microscope for observation, and taking a picture by using a CCD (charge coupled device) to record the dyeing condition of the polyethylene terephthalate plastics in a bright field and a fluorescence state so as to count and calculate the recovery rate of the micro-plastic powder.
Table 2 shows the recovery rates of FITC dye for PE, PS, PVC and PET plastic powders at room temperature, 50 ℃, 75 ℃ and dyeing time of 10min, 20min and 30min
Example 9: process for dyeing polyethylene plastic powder with safranin based on thermal expansion and cold shrinkage method
(1) First, 0.05g of polyethylene plastic powder was weighed into a 250mL beaker, and 49mL of a mixed solution of water and DMSO (volume ratio of 1:9 to 9:1, miscible in any ratio) was added.
(2) The beaker was heated at room temperature, 50 ℃ and 75 ℃ respectively, and after the temperature reached, 1mL of safranin dye (500. Mu.g/mL) was added to start staining and the staining time was recorded.
(3) After the dyeing time reaches 10min, uniformly taking out 10mL of dyeing sample liquid from the beaker, quickly cooling the dyeing sample liquid in the ice-water mixed liquid, and after the dyeing time reaches 20min and 30min, respectively uniformly taking out 10mL of dyeing sample liquid, quickly placing the dyeing sample liquid in the ice-water mixed liquid, and cooling the dyeing sample liquid. Cooling for 5min and taking out.
(4) The cooled dye sample solution was filtered with a teflon filter and washed with a large amount of pure water to wash the dye in the solution.
(5) And finally, putting the filter membrane obtained after suction filtration into 20mL of ultrapure water for ultrasonic treatment to ultrasonically treat the polyethylene plastic powder on the membrane into the pure water, thereby obtaining a sample solution containing the dyed polyethylene plastic powder.
(6) And (3) taking 10 mu L of the sample liquid on a cell counting plate, placing the sample liquid under a fluorescence inverted microscope for observation, and taking a picture by using a CCD (charge coupled device) to record the polyethylene plastic dyeing condition in a bright field and a fluorescence state so as to count and calculate the recovery rate of the micro plastic powder.
Example 10: procedure (1) for dyeing polyvinyl chloride plastic powder with safranin based on the thermal expansion and contraction method 0.05g of polyvinyl chloride plastic powder was first weighed into a 250mL beaker, 49mL of a mixed solution of water and DMSO (in a volume ratio of 1:9 to 9:1, miscible in any ratio) was added.
(2) The beaker was heated at room temperature, 50 ℃ and 75 ℃ respectively, and after the temperature reached, 1mL of safranin dye (500. Mu.g/mL) was added to start staining and the staining time was recorded.
(3) After the dyeing time reaches 10min, uniformly taking out 10mL of dyeing sample liquid from the beaker, quickly cooling the dyeing sample liquid in the ice-water mixed liquid, and after the dyeing time reaches 20min and 30min, respectively uniformly taking out 10mL of dyeing sample liquid, quickly placing the dyeing sample liquid in the ice-water mixed liquid, and cooling the dyeing sample liquid. Cooling for 5min and taking out.
(4) The cooled dye sample solution was filtered with a teflon filter and washed with a large amount of pure water to wash the dye in the solution.
(5) And finally, putting the filter membrane obtained after suction filtration into 20mL of ultrapure water for ultrasonic treatment to ultrasonically treat the polyvinyl chloride plastic powder on the membrane into the pure water, thereby obtaining a sample solution containing the dyed polyvinyl chloride plastic powder.
(6) And (3) taking 10 mu L of the sample liquid on a cell counting plate, placing the sample liquid under a fluorescence inverted microscope for observation, and taking a picture by using a CCD (charge coupled device) to record the polyvinyl chloride plastic dyeing conditions in a bright field and a fluorescence state so as to count and calculate the recovery rate of the micro plastic powder.
Example 11: process for dyeing polystyrene plastic powder with safranin based on thermal expansion and cold shrinkage method
(1) 0.05g of polystyrene plastic powder is initially weighed into a 250mL beaker, and 49mL of a mixed solution of water and DMSO (volume ratio 1:9 to 9:1, miscible in any ratio) are added.
(2) The beaker was heated at room temperature, 50 ℃ and 75 ℃ respectively, and after the temperature reached, 1mL of safranin dye (500. Mu.g/mL) was added to start staining and the staining time was recorded.
(3) After the dyeing time reaches 10min, uniformly taking out 10mL of dyeing sample liquid from the beaker, quickly cooling the dyeing sample liquid in the ice-water mixed liquid, and after the dyeing time reaches 20min and 30min, respectively uniformly taking out 10mL of dyeing sample liquid, quickly placing the dyeing sample liquid in the ice-water mixed liquid, and cooling the dyeing sample liquid. Cooling for 5min and taking out.
(4) The cooled dye sample solution was filtered with a polytetrafluoroethylene filter and washed with a large amount of pure water to wash the dye in the solution.
(5) And finally, putting the filter membrane obtained after suction filtration into 20mL of ultrapure water for ultrasonic treatment so as to ultrasonically treat the polystyrene plastic powder on the membrane into the pure water, thereby obtaining a sample solution containing the dyed polystyrene plastic powder.
(6) And (3) taking 10 mu L of the sample liquid on a cell counting plate, placing the sample liquid under a fluorescence inverted microscope for observation, and taking a picture by using a CCD (charge coupled device) to record the staining condition of the polystyrene plastic in a bright field and a fluorescence state so as to count and calculate the recovery rate of the micro-plastic powder.
Example 12: process for dyeing polyethylene terephthalate plastic powder with safranin based on thermal expansion and cold shrinkage method
(1) 0.05g of polyethylene terephthalate plastic powder is first weighed into a 250mL beaker, and 49mL of a mixed solution of water and DMSO (volume ratio 1:9 to 9:1, miscible in any ratio) are added.
(2) The beaker was left to warm at room temperature, 50 ℃ and 75 ℃ respectively, and after the temperature reached, 1mL of safranin dye (500. Mu.g/mL) was added, staining was started and staining time was recorded.
(3) After the dyeing time reaches 10min, uniformly taking out 10mL of dyeing sample liquid from the beaker, quickly cooling the dyeing sample liquid in the ice-water mixed liquid, and after the dyeing time reaches 20min and 30min, respectively uniformly taking out 10mL of dyeing sample liquid, quickly placing the dyeing sample liquid in the ice-water mixed liquid, and cooling the dyeing sample liquid. Cooling for 5min and taking out.
(4) The cooled dye sample solution was filtered with a teflon filter and washed with a large amount of pure water to wash the dye in the solution.
(5) And finally, putting the filter membrane obtained after suction filtration into 20mL of ultrapure water for ultrasonic treatment to ultrasonically treat the polyethylene terephthalate plastic powder on the membrane into the pure water, thereby obtaining a sample solution containing the dyed polyethylene terephthalate plastic powder.
(6) And (3) taking 10 mu L of the sample solution, placing the sample solution on a cell counting plate, observing the sample solution under a fluorescence inverted microscope, and taking a CCD picture to record the staining condition of the polyethylene terephthalate plastics in a bright field and a fluorescence state so as to count and calculate the recovery rate of the micro-plastic powder.
Table 3 shows the recovery rates of the micro plastic powder in the physical dyeing method of the safranin dye for four plastic powders of PE, PS, PVC and PET at room temperature, 50 ℃, 75 ℃ and the dyeing time of 10min, 20min and 30min
According to the invention, the recovery rates of the four plastic powders of PE, PS, PVC and PET are respectively carried out on three dyes (Nile red, FITC and safranin) under the conditions of 25 ℃, 50 ℃, 75 ℃ and 10min, 20min and 30min of dyeing time. As can be seen from FIGS. 1 to 3, the PE, PS, PVC and PET plastic powders dyed with the three dyes have strong fluorescence signals and are easy to identify. As can be seen from tables 1 to 3, the recovery rates of PE, PVC, PS and PET of the plastic powders dyed with the three dyes are mostly greater than 95% at any temperature and dyeing time, which indicates that the three dyes have significant coloring effects on the four plastic powders.
The invention utilizes the physical characteristics of plastic of expansion with heat and contraction with cold, selects three dyes of Nile red, fluorescein Isothiocyanate (FITC) and safranine to dye four common plastic powders (polyethylene, polystyrene, polyvinyl chloride and polyethylene terephthalate) in the environment under different heating conditions. The results show that the four plastic powders are easily colored by the three dyes, and according to the experimental dyeing results, the optimal dyeing conditions of each plastic are confirmed, and the effect of dyeing the polyethylene plastic powder by safranin for 20min at the temperature of 50 ℃ is the best; for polystyrene plastic powder it is most suitable to dye with FITC dye at 50 ℃ for 20 min; and the polyethylene terephthalate and polyvinyl chloride plastic powder has better dyeing effect for 20min by using NR dye at 25 ℃. According to the observation of the retention time of the plastic powder after dyeing for two months, the fluorescence intensity of the plastic powder dyed by the physical and chemical methods is found to be as strong as that before when the plastic powder is observed after two months, and no obvious difference exists, which indicates that the plastic powder dyed by the physical dyeing method has the same stability as that of the plastic powder dyed by the chemical method.
Further, in the present invention, in order to verify the effectiveness of the physical staining method, a chemical bonding staining method was compared, which is based on that an amino group on one end of a silane reagent may be bonded to an isothiocyanato group on FITC dye, and a siloxy group on the other end may be bonded to a hydroxyl group on plastic, thereby allowing the plastic to be fluorescently labeled.
Comparative example: chemical bonding dyeing method
To verify the effectiveness of the physical staining method, the chemical bonding staining method was compared, which is based on that the amino group on one end of the silane reagent can bond with the isothiocyanato group on the FITC dye, while the siloxy group on the other end can bond with the hydroxyl group on the plastic, thereby making the plastic fluorescently labeled. The method comprises the following specific steps:
(1) First, 20mL of a silane reagent (trimethoxy silane) and a methanol (1: 19) solution were weighed and mixed in a beaker, and then 0.02g of plastic powder was weighed and added into the beaker containing the mixed solution for a silylation reaction for 12 hours.
(2) After the silanization reaction is finished, the solution in the beaker is filtered and washed by a polytetrafluoroethylene filter membrane.
(3) And (3) putting the filter membrane after suction filtration into 49mL of methanol for ultrasonic treatment to ultrasonically treat the micro plastic powder on the membrane into the methanol solution, thereby obtaining a plastic powder sample solution after the silanization reaction.
(4) To the above plastic powder sample solution was added 1mL of FITC (500. Mu.g/mL) dye and the mixture was stained for 3 hours.
(5) And then, carrying out suction filtration and washing on the dyed sample liquid by using a polytetrafluoroethylene filter membrane until the solution is colorless, and then putting the filter membrane obtained after suction filtration into 20mL of ultrapure water for ultrasonic treatment so as to carry out ultrasonic treatment on the micro-plastic powder on the membrane into the pure water, thereby obtaining a sample liquid containing the dyed micro-plastic powder.
(6) And (3) taking 10 mu L of the sample liquid on a cell counting plate, placing the sample liquid under a fluorescence inverted microscope for observation, and taking a picture by using a CCD (charge coupled device) to record the micro-plastic conditions in a bright field and a fluorescence state so as to count and calculate the recovery rate of the micro-plastic powder.
From the fluorescent signal condition of the micro plastic powder after two months in the two dyeing methods, the sample dyed by the physical and chemical methods is kept for two months, and the fluorescent condition is respectively observed after two months. As shown in fig. 5 to 6, there is no significant difference in fluorescence intensity after two months ago for the four plastic powders in the physical dyeing method and the chemical dyeing method, which is probably because the dye molecules bonded to the plastic surface in the chemical dyeing method are relatively stable, whereas the dye molecules in the physical method are wrapped in the plastic and are not easily dropped.
Test examples
Test example 1: recovery of micro-plastic powder
The recovery rates of the three dyes (Nile red, FITC and safranin) on four plastic powders of PE, PS, PVC and PET are respectively carried out at 25 ℃, 50 ℃, 75 ℃ and 10min, 20min and 30min of dyeing time. As can be seen from Table 1, the recovery rates of PE, PVC, PS and PET of the four plastic powders are more than 95% regardless of the temperature and dyeing time of the four plastic powders dyed with Nile Red, which indicates that the coloring effect of the Nile Red on the four plastic powders is very significant.
Test example 2: raman spectroscopy of micro-plastic powders
As can be seen from fig. 8, the raman spectra of the four types of micro plastic powders at 75 ℃ were not significantly different from those of the micro plastic standard without any treatment, indicating that the four types of micro plastic powders were hardly affected at 75 ℃. And comparing the Raman spectra of the four types of micro plastic powder before and after being dyed with Nile Red, FITC and safranin respectively at 75 ℃, almost no difference can be seen, which indicates that the three types of dyes have no destructive effect on the micro plastic powder.
According to the observation of the retention time of the plastic powder after dyeing for two months, the fluorescence intensity of the plastic powder dyed by the physical and chemical methods is found to be as strong as that before when the plastic powder is observed after two months, and no obvious difference exists, which indicates that the plastic powder dyed by the physical dyeing method has the same stability as that of the plastic powder dyed by the chemical method. But because the chemical staining method is long in time and the required reagents are expensive, the method is simpler and quicker compared with a physical staining method based on expansion and contraction, the sample retention time is long, and the method is not limited to the selection of dyes and the type of micro-plastics, and provides a reliable method for identifying the micro-plastics in the environment.
The four plastic powders of PE, PS, PVC and PET are easily colored by dye under the heating state. And according to the dyeing result, the optimal dyeing conditions of each plastic powder type are confirmed as follows: the best effect is achieved when the PE plastic powder is dyed with safranin for 20min at the temperature of 50 ℃; for PS plastic powder, dyeing with FITC dye at 50 ℃ for 20min is most suitable; the PET and PVC plastic powder has better dyeing effect for 20min by using NR dye at 25 ℃. According to the observation of the retention time of the plastic powder after dyeing for two months, the fluorescence intensity of the plastic powder dyed by the physical and chemical methods is found to be as strong as that before when the plastic powder is observed after two months, and no obvious difference exists, which indicates that the plastic powder dyed by the physical dyeing method has the same stability as that of the plastic powder dyed by the chemical method. But because the chemical staining method is longer in time and the required reagent is expensive, the method is simpler and quicker compared with a physical staining method based on expansion and contraction, the sample retention time is long, and the method is not limited by the selection of dyes and the type of micro-plastics, and the method provides a reliable method for identifying the micro-plastics in the environment.
Claims (10)
1. A fluorescent staining and marking method of micro plastic is characterized in that: the method is characterized in that the micro plastic is subjected to fluorescent dyeing marking by adopting a physical method based on the characteristic of expansion with heat and contraction with cold, and the coloring step of the micro plastic powder is that the micro plastic powder selected from Polyethylene (PE), polystyrene (PS), polyvinyl chloride (PVC) and polyethylene terephthalate (PET) is added into the micro plastic powder in a volume ratio of 1:9 to 9:1, water and DMSO mixed solution which is miscible in any proportion; heating at room temperature to 75 ℃, adding a dye solution for dyeing when the temperature reaches 50 ℃ or 75 ℃, quickly cooling in an ice-water mixed solution for 5 minutes after dyeing for 10 to 30 minutes, and taking out.
2. The method for fluorescent staining and marking of a micro-plastic according to claim 1, wherein: the method comprises the following steps:
micro plastic is added into the mixture in a volume ratio of 1:9 to 9:1, the mixed solution of water and DMSO miscible in any proportion expands under heating;
and adding a dye solution into the mixed solution so as to facilitate the dye solution to enter the interior of the micro plastic, heating for a specified time, rapidly cooling in the ice-water mixed solution for 5min, and taking out to lock the dye in the interior of the plastic, so that the micro plastic is marked by the fluorescent dye.
3. The method for fluorescent staining and marking of a micro-plastic according to claim 1, wherein: the micro plastic comprises at least one of four plastic powders of Polyethylene (PE), polystyrene (PS), polyvinyl chloride (PVC) and polyethylene terephthalate (PET).
4. The method for fluorescent staining and marking of a micro-plastic according to claim 1, wherein: the heating temperature of the mixed solution is selected from any temperature range of 25-75 ℃.
5. The method for fluorescent staining and marking of a micro plastic according to claim 1, wherein: the dyeing time is any time from 10 minutes to 30 minutes.
6. The method for fluorescent staining and marking of a micro-plastic according to claim 1, wherein: the dye solution is selected from Nile Red (NR) dye, fluorescein Isothiocyanate (FITC) or safranin dye.
7. A fluorescent staining labeling method for quantifying micro-plastics is characterized in that: the method comprises the following steps: preparing a dye solution, coloring the micro plastic powder, filtering, and calculating the recovery rate of the micro plastic powder, wherein the coloring step of the micro plastic powder is to add the micro plastic powder selected from Polyethylene (PE), polystyrene (PS), polyvinyl chloride (PVC) and polyethylene terephthalate (PET) into the dye solution in a volume ratio of 1:9 to 9:1, water and DMSO mixed solution which is miscible in any proportion; heating at room temperature to 75 ℃, adding a dye solution for dyeing when the temperature reaches 50 ℃ or 75 ℃, quickly cooling in an ice-water mixed solution for 5 minutes after dyeing for 10 to 30 minutes, and taking out.
8. The fluorescent staining labeling method of quantitative microplastics according to claim 7, characterized in that:
the dye solution is selected from a Nile Red (NR) dye dissolved in dimethyl sulfoxide (DMSO) to prepare a Nile red dye solution with the concentration of 500 mu g/mL, or a Fluorescein Isothiocyanate (FITC) and a safranin dye dissolved in pure water to prepare a dye solution with the concentration of 500 mu g/mL.
9. The fluorescent staining labeling method of quantitative microplastics according to claim 7, characterized in that:
and the step of suction filtration is that the cooled sample liquid is suction filtered by a polytetrafluoroethylene filter membrane, washed by a small amount of DMSO solution to remove redundant dye solution, and washed by pure water until the solution is colorless.
10. The fluorescent staining method for quantifying a micro plastic according to claim 7, wherein:
the step of calculating the recovery rate of the micro-plastic powder comprises the steps of placing the cell counting plate under a fluorescence inverted microscope for observation, and photographing and recording the micro-plastic conditions in a bright field and a fluorescence state by using a CCD (charge coupled device) so as to count and calculate the recovery rate of the micro-plastic powder.
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