CN115753720A - Micro-plastic fluorescent staining quantitative detection method based on Nile red staining agent - Google Patents
Micro-plastic fluorescent staining quantitative detection method based on Nile red staining agent Download PDFInfo
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Abstract
The invention relates to a quantitative detection method for fluorescent staining of micro-plastics based on a Nile red staining agent, which comprises the steps of putting a pollutant sample into a methylene blue working solution for oscillation reaction, and then putting the pollutant sample into a Nile red staining agent working solution for water bath heating reaction, so that the problem of co-staining of Nile red and natural organic matters and biomass in the pollutant sample can be avoided, and false positive can be prevented, and the problem of larger quantitative result caused by the single use of the Nile red staining agent can be effectively solved; the novel fluorescent dyeing method formed by the combined action of the methylene blue dye solution and the nile red dye solution can avoid the problem of common dyeing of natural organic matters in the process of measuring the micro-plastic, the measurement result is more accurate, the heating and cooling circulation in the dyeing process can effectively enhance the dyeing effect, the fluorescence intensity can be kept for a longer time, the fluorescence quenching caused by acetone volatilization is avoided, the clear and bright fluorescence effect is obtained, and the detection reliability is higher.
Description
Technical Field
The invention relates to the technical field of detection of micro-plastic content, in particular to a micro-plastic fluorescent staining quantitative detection method based on a Nile red staining agent.
Background
Microplastics generally refer to a new class of artificial pollutants with a particle size of less than 5 mm, which gradually release artificial additives during slow degradation. Because the particle size is small and the hydrophobicity is strong, the micro plastic is easy to be attached to organic pollutants and microorganisms in the environment to form composite pollutants, and after being ingested by aquatic organisms, the toxicity can be transferred and enriched in a food chain, thereby causing harm to aquatic ecosystems and even human health. The abundance of the micro-plastic is an important index for judging the pollution degree of the micro-plastic. The size and the mass of the micro-plastic are small, so that if the abundance is quantified from the mass perspective, the mass fraction of the micro-plastic, namely the content of the micro-plastic in a unit mass sample, can only be determined by adopting a thermal analysis method in the prior art; if the abundance is quantified quantitatively, the technical classification statistics of a combined microscope is adopted.
After the links of sample collection, digestion, density separation and quality control, the micro-plastic which is difficult to be detected by naked eyes can be marked by a fluorescence method so as to enhance the objectivity of identification and conveniently and quickly carry out micro-plastic quantification. The Nile red dyeing fluorescence method is considered to have wide prospect in the aspect of rapidly and effectively identifying the micro-plastic, and as a lipophilic dye, green fluorescence can be adopted to carry out fluorescence microscopy on the micro-plastic after Nile red dyeing. However, in the practical use of nile red, the natural organic matters and the biomass in the water body inevitably generate a certain co-dyeing problem, so that the generation of 'false positive' is caused.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a micro-plastic fluorescent staining quantitative detection method based on a Nile red staining agent, which is simple in determination method and can effectively solve the problem of large quantitative result caused by single use of the Nile red staining agent.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows: a micro-plastic fluorescent staining quantitative detection method based on a Nile red staining agent comprises the following steps:
step S1, preparing a methylene blue dye solution and a nile red dye solution respectively to obtain a methylene blue working solution and a nile red dye working solution;
s2, acquiring a pollutant sample containing the micro-plastics, and pretreating the pollutant sample to remove the microbial biomass to obtain a pollutant sample from which the microbial biomass is removed;
s3, adding a pollutant sample for removing the microbial biomass into the methylene blue working solution, and carrying out oscillation reaction to obtain a first reaction solution;
s4, adding the working solution of the Nile red dye into the first reaction solution, and heating in a water bath for reaction to obtain a second reaction solution;
and S5, cooling the second reaction liquid to room temperature, carrying out vacuum filtration, retaining the dyed micro-plastic, observing a fluorescent substance by using a fluorescence microscope at the wavelength of 520-530 nm, counting, measuring the size, and determining the content of the micro-plastic in the pollutant sample.
Further, in the step S1, a methylene blue reagent is added into the aqueous solution to prepare a methylene blue working solution with the concentration of 0.1% -1%; adding a biological stain Nile Red into acetone for dissolving, and diluting with a normal hexane solution to prepare a Nile Red dye working solution with the concentration of 1-20 mu g/mL.
Further, in the step S1, a methylene blue reagent is added into the aqueous solution to prepare a methylene blue working solution with a concentration of 0.6% -1%; adding a biological stain Nile Red into acetone for dissolving, and diluting by using a normal hexane solution to prepare a Nile Red dye working solution with the concentration of 10-20 mu g/mL.
Further, the contaminant sample containing the micro plastic in the step S2 is selected from a water sample, an air sample or a solid sample; the weight of the sample to be measured is 1 mug-1 kg.
Further, the solid sample in step S2 is selected from the group consisting of soil, food intermediate, compost, sludge, sediment, biological sample, and animal waste.
Further, the biological sample in step S2 is selected from an animal or a plant, an organ of an animal, a tissue of a plant or a sample of animal stomach contents.
Further, in the step S3, a pollutant sample from which the microbial biomass is removed is added to the methylene blue working solution, and the mixture is subjected to oscillation reaction at room temperature for 5 to 10min to obtain a first reaction solution; the volume ratio of the contaminant sample for removing the microbial biomass to the methylene blue working solution is (100).
Further, the nile red dye working solution is added into the first reaction solution in the step S4, and the mixture is heated in a water bath at 35-70 ℃ for reaction for 10-15 min to obtain a second reaction solution; the volume ratio of the contaminant sample for removing the microbial biomass to the Nile Red dye working solution is (100).
Further, in the step S5, the second reaction solution is cooled to room temperature, a wharman filter membrane is used for vacuum filtration, the dyed micro-plastic is retained, the filtered organic membrane is placed in a clean culture dish, a fluorescent substance is observed by using a fluorescence microscope at a wavelength of 520-530 nm, counting and sizing are performed, and the content of the micro-plastic in the pollutant sample is determined.
Further, the pore size of the Whanman filter in step S5 is 1.2 μm.
The invention has the beneficial effects that: in the actual use of Nile red, a certain co-dyeing problem inevitably occurs between natural organic matters and biomass in a water body, so that 'false positive' is generated; the novel fluorescent dyeing method formed by the combined action of the methylene blue dye solution and the nile red dye solution can avoid the problem of common dyeing of natural organic matters in the process of measuring the micro-plastic, the measurement result is more accurate, the heating and cooling circulation in the dyeing process can effectively enhance the dyeing effect, the fluorescence intensity can be kept for a longer time, the fluorescence quenching caused by acetone volatilization is avoided, the clear and bright fluorescence effect is obtained, and the detection reliability is higher.
Detailed Description
The following examples may help one skilled in the art to more fully understand the present invention, but are not intended to limit the invention in any way.
The invention relates to a fluorescent staining quantitative detection method for micro-plastics based on a Nile red staining agent, wherein an environmental sample is selected from a water sample, an air sample or a solid sample; the solid sample is selected from soil, food intermediate products, compost, sludge, sediment, biological samples or animal waste and the like, but the solid sample is not limited to the above substances and can also be sand and stone and the like, and the solid sample can be selected variously according to actual needs; the biological sample is selected from the group consisting of an animal, a plant, an organ of an animal, a tissue of a plant, a content sample of an animal stomach, and the like, but is not limited thereto, and may be animal blood, and the like.
The invention relates to a quantitative detection method for fluorescent staining of micro-plastics based on a Nile red staining agent, wherein the mass of a detected sample to be detected can be between 1 mu g and 1kg, specifically between 1mg and 10g, or increased to the mass between 1mg and 30 mg.
The invention relates to a quantitative detection method for fluorescent staining of micro-plastic based on a Nile red staining agent, which is methylene blue working solution with the concentration of 0.1-1%, in particular to methylene blue in the methylene blue working solution with the mass fraction of 0.1-1%.
The invention relates to a micro-plastic fluorescent staining quantitative detection method based on a Nile red staining agent, which comprises the following steps of:
1. preparing a methylene blue dye solution and a nile red dye solution respectively to obtain a methylene blue working solution and a nile red dye working solution;
2. obtaining a pollutant sample containing micro-plastics, wherein the weight of the pollutant sample is 1 microgram-1 kg, and pretreating the pollutant sample to remove microbial biomass to obtain the pollutant sample without the microbial biomass;
3. adding a pollutant sample for removing the microbial biomass into the methylene blue working solution, and performing oscillation reaction to obtain a first reaction solution;
4. adding the Nile red dye working solution into the first reaction solution, and heating in a water bath for reaction to obtain a second reaction solution;
5. and cooling the second reaction liquid to room temperature, carrying out vacuum filtration, retaining the dyed micro-plastic, observing a fluorescent substance by using a fluorescence microscope at the wavelength of 520-530 nm, counting, measuring the size, and determining the content of the micro-plastic in the pollutant sample.
Example 1
A micro-plastic fluorescent staining quantitative detection method based on a Nile red staining agent comprises the following steps:
step S1, adding a methylene blue reagent into an aqueous solution at room temperature to prepare a methylene blue working solution with the concentration of 0.1%, and covering and storing the methylene blue working solution by using an aluminum foil after the preparation is finished; adding a biological stain Nile red into a small amount of acetone for dissolving, and diluting with a normal hexane solution to prepare a Nile red dye working solution with the concentration of 1 mu g/mL, wherein the Nile red dye working solution is covered and stored by an aluminum foil after being prepared;
s2, 20 g of a pollutant sample containing the micro-plastics is obtained, the sample is selected from soil, chloroform is used for soaking the pollutant sample to remove a certain amount of microbial biomass, then cleaning is carried out, finally, high-density zinc bromide is used for separating the micro-plastics into a supernatant, and the supernatant is obtained by separation, wherein the supernatant is the pollutant sample from which the microbial biomass is removed;
s3, adding a pollutant sample for removing the microbial biomass into the methylene blue working solution, and carrying out oscillation reaction for 5min at room temperature (25 ℃) to obtain a first reaction solution; the volume ratio of the pollutant sample for removing the microbial biomass to the methylene blue working solution is 100;
s4, adding the working solution of the Nile red dye into the first reaction solution, and heating in a water bath at 35 ℃ for 10min to react to obtain a second reaction solution; the volume ratio of the contaminant sample with the microbial biomass removed to the Nile Red dye working solution is 100;
step S5, performing vacuum filtration on the second reaction solution by using a Whanman filter membrane, reserving the dyed micro-plastics, placing the filtered organic membrane in a clean culture dish, observing fluorescent substances at the wavelength of 520-530 nm by using a fluorescence microscope, counting, measuring the size, and determining the content of the micro-plastics in the pollutant sample; the pore size of the Whanman filter was 1.2. Mu.m.
Example 2
A micro-plastic fluorescent staining quantitative detection method based on a Nile red staining agent comprises the following steps:
step S1, adding a methylene blue reagent into an aqueous solution at room temperature to prepare a methylene blue working solution with the concentration of 1%, and covering and storing the methylene blue working solution with aluminum foil after the preparation is finished; adding a biological stain Nile red into a small amount of acetone for dissolving, and diluting with a normal hexane solution to prepare a Nile red dye working solution with the concentration of 20 mu g/mL, wherein the Nile red dye working solution is covered and stored by an aluminum foil after being prepared;
s2, obtaining 200 milliliters of a pollutant sample containing the micro-plastics, wherein the sample is selected from river water, soaking the pollutant sample by using dilute nitric acid to remove a certain amount of microbial biomass, then cleaning, finally separating the micro-plastics to supernatant by using high-density zinc bromide, and separating the supernatant to obtain supernatant, wherein the supernatant is the pollutant sample from which the microbial biomass is removed;
s3, adding a pollutant sample for removing the microbial biomass into the methylene blue working solution, and performing oscillation reaction for 10min at room temperature to obtain a first reaction solution; the volume ratio of the microbial biomass removal pollutant sample to the methylene blue working solution is 100;
s4, adding the working solution of the Nile red dye into the first reaction solution, and heating in a water bath at 70 ℃ for 15min to react to obtain a second reaction solution; the volume ratio of the pollutant sample for removing the microbial biomass to the Nile red dye working solution is 100;
step S5, performing vacuum filtration on the second reaction solution by using a Whanman filter membrane, reserving the dyed micro-plastics, placing the filtered organic membrane in a clean culture dish, observing fluorescent substances at the wavelength of 520-530 nm by using a fluorescence microscope, counting, measuring the size, and determining the content of the micro-plastics in the pollutant sample; the pore size of the Whanman filter was 1.2. Mu.m.
Example 3
A micro-plastic fluorescent staining quantitative detection method based on a Nile red staining agent comprises the following steps:
step S1, adding a methylene blue reagent into an aqueous solution at room temperature to prepare a methylene blue working solution with the concentration of 0.6%, and covering and storing the methylene blue working solution by using an aluminum foil after the preparation is finished; adding a biological stain Nile red into a small amount of acetone for dissolving, and diluting with a normal hexane solution to prepare a Nile red dye working solution with the concentration of 10 mu g/mL, wherein the Nile red dye working solution is covered and stored by an aluminum foil after being prepared;
s2, obtaining a pollutant sample 20 mg containing the micro-plastics, wherein the sample is selected from sludge, soaking the pollutant sample by using dilute nitric acid to remove a certain amount of microbial biomass, then cleaning, finally separating the micro-plastics to supernatant by using high-density zinc bromide, and separating the supernatant to obtain supernatant, wherein the supernatant is the pollutant sample from which the microbial biomass is removed;
s3, adding a pollutant sample for removing the microbial biomass into the methylene blue working solution, and performing oscillation reaction for 8min at room temperature to obtain a first reaction solution; the volume ratio of the microbial biomass removal pollutant sample to the methylene blue working solution is 100;
s4, adding the Nile red dye working solution into the first reaction solution, and heating in a water bath at 60 ℃ for reaction for 12min to obtain a second reaction solution; the volume ratio of the contaminant sample with the microbial biomass removed to the Nile Red dye working solution is 100;
step S5, performing vacuum filtration on the second reaction solution by using a Whanman filter membrane, reserving the dyed micro-plastics, placing the filtered organic membrane in a clean culture dish, observing fluorescent substances at the wavelength of 520-530 nm by using a fluorescence microscope, counting, measuring the size, and determining the content of the micro-plastics in the pollutant sample; the pore size of the Whanman filter was 1.2. Mu.m.
Comparative example 1
A micro-plastic fluorescent staining quantitative detection method based on a Nile red staining agent comprises the following steps:
step S1, adding a methylene blue reagent into an aqueous solution at room temperature (25 ℃) to prepare a methylene blue working solution with the concentration of 0.6%, and covering and storing the methylene blue working solution by using an aluminum foil after the preparation is finished; adding a biological stain Nile Red into a small amount of acetone for dissolving, diluting with a normal hexane solution to prepare a Nile Red dye working solution with the concentration of 10 mu g/mL, and covering and storing the Nile Red dye working solution with an aluminum foil after the preparation is finished;
s2, obtaining a pollutant sample containing micro-plastics 20 mg, wherein the sample is selected from sludge, soaking the pollutant sample by using dilute nitric acid to remove a certain amount of microbial biomass, then cleaning, finally separating the micro-plastics to supernate by using high-density zinc bromide, and separating to obtain supernate, wherein the supernate is the pollutant sample from which the microbial biomass is removed;
s3, adding a pollutant sample for removing the microbial biomass into the methylene blue working solution, and carrying out oscillation reaction for 8min at room temperature (25 ℃) to obtain a first reaction solution; the volume ratio of the pollutant sample for removing the microbial biomass to the methylene blue working solution is 100;
s4, adding the working solution of the Nile red dye into the first reaction solution, and reacting for 12min at room temperature (25 ℃) to obtain a second reaction solution; the volume ratio of the contaminant sample with the microbial biomass removed to the Nile Red dye working solution is 100;
step S5, performing vacuum filtration on the second reaction solution by using a Whanman filter membrane, reserving the dyed micro-plastics, placing the filtered organic membrane in a clean culture dish, observing fluorescent substances at the wavelength of 520-530 nm by using a fluorescence microscope, counting, measuring the size, and determining the content of the micro-plastics in the pollutant sample; the pore size of the Whanman filter was 1.2. Mu.m.
Comparative example 2
A micro-plastic fluorescent staining quantitative detection method based on a Nile red staining agent comprises the following steps:
step S1, adding a biological stain Nile Red into a small amount of acetone at room temperature for dissolving, diluting with a normal hexane solution to prepare a Nile Red dye working solution with the concentration of 10 mu g/mL, and covering and storing the Nile Red dye working solution with an aluminum foil after the preparation is finished;
s2, obtaining a pollutant sample 20 mg containing the micro-plastics, wherein the sample is selected from sludge, soaking the pollutant sample by using dilute nitric acid to remove a certain amount of microbial biomass, then cleaning, finally separating the micro-plastics to supernatant by using high-density zinc bromide, and separating the supernatant to obtain supernatant, wherein the supernatant is the pollutant sample from which the microbial biomass is removed;
s3, adding a pollutant sample from which the microbial biomass is removed into the working solution of the Nile red dye, and heating in a water bath at 60 ℃ for 12min to react to obtain a first reaction solution; the volume ratio of the contaminant sample with the microbial biomass removed to the Nile Red dye working solution is 100;
s4, performing vacuum filtration on the first reaction solution by using a Whanman filter membrane, reserving the dyed micro-plastics, placing the filtered organic membrane in a clean culture dish, observing fluorescent substances at the wavelength of 520-530 nm by using a fluorescence microscope, counting, measuring the size, and determining the content of the micro-plastics in the pollutant sample; the pore size of the Whanman filter was 1.2. Mu.m.
The experimental results of the above examples 1 to 3 and comparative examples 1 to 2 are shown in the following table 1:
TABLE 1
| Item | Whether the co-staining results in "false positives" | Whether or not to result in a greater quantitative result | Correlation coefficient R2 of standard curve | Fluorescent effect | Fluorescence intensity at day 7 is the initial fluorescence intensity |
| Example 1 | Whether or not | Whether or not | 0.996 | Clear and bright fluorescent effect | 98.5%-99.5% |
| Example 2 | Whether or not | Whether or not | 0.997 | Very clear and bright fluorescent effect | 98.5%-99.5% |
| Example 3 | Whether or not | Whether or not | 0.998 | Very clear and bright fluorescent effect | 98.5%-99.5% |
| Comparative example 1 | Whether or not | Whether or not | 0.985 | Generally clear and bright fluorescent effect | 84.5%-88.5% |
| Comparative example 2 | Is that | Is that | 0.952 | Generally clear and bright fluorescent effect | 73.5% or less |
According to the experimental results of the embodiments 1-3 and the comparative examples 1-2, the fluorescent staining quantitative detection method for the micro-plastics based on the Nile Red coloring agent, disclosed by the invention, comprises the steps of putting a pollutant sample into a methylene blue working solution for oscillation reaction, then putting the pollutant sample into a Nile Red dye working solution for water bath heating reaction, wherein the methylene blue is used as an auxiliary dye, so that most of organic matters and biomass can be dyed, the problem of certain co-staining of the Nile Red and natural organic matters and biomass in the pollutant sample is avoided, the generation of false positive is prevented, the problem of larger quantitative result caused by the single use of the Nile Red dye can be effectively solved, the operation is simple and convenient, the error is small, and the detection sensitivity is high; the novel fluorescent dyeing method formed by the combined action of the methylene blue dye solution and the nile red dye solution can avoid the problem of common dyeing of natural organic matters in the process of measuring the micro-plastic, the measurement result is more accurate, the heating and cooling circulation in the dyeing process can effectively enhance the dyeing effect, the fluorescence intensity can be kept for a longer time, the fluorescence quenching caused by acetone volatilization is avoided, the clear and bright fluorescence effect is obtained, and the detection reliability is higher.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.
Claims (10)
1. A micro-plastic fluorescent staining quantitative detection method based on a Nile red staining agent is characterized by comprising the following steps:
step S1, preparing a methylene blue dye solution and a nile red dye solution respectively to obtain a methylene blue working solution and a nile red dye working solution;
s2, obtaining a pollutant sample containing the micro-plastics, and pretreating the pollutant sample to remove the microbial biomass to obtain the pollutant sample without the microbial biomass;
s3, adding a pollutant sample for removing the microbial biomass into the methylene blue working solution, and performing oscillation reaction to obtain a first reaction solution;
s4, adding the working solution of the Nile red dye into the first reaction solution, and heating in a water bath for reaction to obtain a second reaction solution;
and S5, cooling the second reaction liquid to room temperature, carrying out vacuum filtration, retaining the dyed micro-plastic, observing a fluorescent substance by using a fluorescence microscope at the wavelength of 520-530 nm, counting, measuring the size, and determining the content of the micro-plastic in the pollutant sample.
2. The method for quantitative detection of fluorescent staining of micro-plastic based on Nile red staining agent of claim 1, wherein in step S1, methylene blue reagent is added into aqueous solution to prepare methylene blue working solution with concentration of 0.1% -1%; adding a biological stain Nile Red into acetone for dissolving, and diluting with a normal hexane solution to prepare a Nile Red dye working solution with the concentration of 1-20 mu g/mL.
3. The method for quantitatively detecting the fluorescent staining of the micro plastic based on the nile red staining agent as claimed in claim 2, wherein the methylene blue reagent is added into the aqueous solution in the step S1 to prepare a methylene blue working solution with a concentration of 0.6 to 1 percent; adding a biological stain Nile Red into acetone for dissolving, and diluting by using a normal hexane solution to prepare a Nile Red dye working solution with the concentration of 10-20 mu g/mL.
4. The method for quantitatively detecting the fluorescent staining of the micro plastic based on the nile red staining agent of claim 1, wherein the contaminant sample containing the micro plastic in the step S2 is selected from a water sample, an air sample or a solid sample; the weight of the sample to be measured is 1 mug-1 kg.
5. The method for quantitative detection of fluorescent staining of micro-plastics based on Nile Red staining agent of claim 4, wherein the solid sample in step S2 is selected from soil, food, intermediate products of food, compost, sludge, sediment, biological sample or animal waste.
6. The method for quantitative detection of fluorescent staining of micro plastic based on Nile Red stain of claim 4, wherein the biological sample in step S2 is selected from the group consisting of animal or plant, animal organ, plant tissue and animal stomach content sample.
7. The method for quantitative detection of fluorescent staining of micro-plastic based on Nile Red staining agent of claim 1, wherein in the step S3, a contaminant sample from which a microbial substance is removed is added to the methylene blue working solution, and the mixture is subjected to oscillation reaction at room temperature for 5-10 min to obtain a first reaction solution; the volume ratio of the contaminant sample for removing the microbial biomass to the methylene blue working solution is (100).
8. The method for quantitative detection of fluorescent staining of micro-plastic based on Nile red staining agent of claim 1, wherein the Nile red dye working solution is added into the first reaction solution in the step S4, and the reaction is carried out in a water bath at 35-70 ℃ for 10-15 min to obtain a second reaction solution; the volume ratio of the contaminant sample for removing the microbial biomass to the working solution of the Nile Red dye is (100).
9. The method as claimed in claim 1, wherein in step S5, the second reaction solution is cooled to room temperature, and vacuum filtered by a wharman filter membrane, the dyed micro-plastics are retained, the filtered organic membrane is placed in a clean culture dish, fluorescent substances are observed by a fluorescence microscope at a wavelength of 520-530 nm, and the content of the micro-plastics in the pollutant sample is determined by counting and measuring the size.
10. The method for quantitative detection of fluorescent staining of micro-plastics based on Nile red stain as claimed in claim 9, wherein the pore size of Whanman filter membrane in step S5 is 1.2 μm.
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| CN117007570A (en) * | 2023-10-08 | 2023-11-07 | 海南热带海洋学院 | Method for detecting microplastic in marine organism |
| CN117074376A (en) * | 2023-07-07 | 2023-11-17 | 广东海洋大学 | Method for quenching fluorescence in microplastic and method for detecting microplastic |
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| CN117074376A (en) * | 2023-07-07 | 2023-11-17 | 广东海洋大学 | Method for quenching fluorescence in microplastic and method for detecting microplastic |
| CN117007570A (en) * | 2023-10-08 | 2023-11-07 | 海南热带海洋学院 | Method for detecting microplastic in marine organism |
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