A method for quantitatively detecting microplastics in water based on Nile red staining - cell-like counting

Abstract

The present invention discloses a method for quantitatively detecting microplastics in water based on Nile red staining - cell-like counting, comprising the following steps: (1) preparing a standard water sample with microplastics having different particle size ranges and known particle sizes, subjecting the standard water sample to suction filtration, elution, digestion, and staining using a filter membrane; detecting the stained water sample by a flow cytometer to obtain the FSC channel signal intervals of microplastics with different particle size ranges; (2) subjecting the water sample to be detected to suction filtration, elution, digestion, and staining using a filter membrane; detecting the stained water sample by a flow cytometer, and respectively counting the microplastics with different particle size ranges according to the FSC channel signal intervals of microplastics with different particle size ranges obtained in step (1). The quantitative detection method of the present invention can effectively eliminate the interference of other impurity particles on the detection result, and automatically count the small particle size microplastic particles with a size of 1 - 100 μm in different zones.

Description

Technical Field

[0001] The present invention relates to the technical field of detection of new pollutants in the water environment, and particularly relates to a method for quantitatively detecting microplastics in water based on Nile red staining and cell-like counting. Background Art

[0002] Due to the substantial increase in the global consumption of plastic products and the poor management of plastic waste, microplastics with a diameter of less than 5 mm have become one of the new pollutants attracting much attention. Over time, small-sized microplastics (1 - 100 μm) have gradually become the end destination of plastic products, and their abundance in the environment will continue to increase. The smaller the particle size, the faster the particle abundance increases, and ultimately they accumulate in organisms. In addition, compared with larger-sized microplastics, small-sized microplastics may have stronger environmental stability, are more likely to become carriers of other pollutants and be colonized by microorganisms, bringing higher environmental exposure risks and biological ingestion levels.

[0003] Currently, the main detection methods for small-sized microplastics include spectroscopy, chromatography, microscopy, etc. Using spectroscopy to identify each individual microplastic particle has a long detection time and high detection costs, and the smaller the particle, the stronger the background interference. When the particle diameter is less than 5 μm, the accuracy of spectroscopy is greatly reduced. Chromatography quantitatively analyzes the mass concentration of microplastics by means of destruction, but cannot obtain information such as the size and shape of the polymer. Visual inspection of small-sized microplastics through a microscope is not ideal, and subjective factors can lead to underestimation or overestimation of the results. For the detection of small-sized microplastics in the water environment, the above common detection methods are difficult to achieve a good balance among precision, accuracy, efficiency, and economy.

[0004] Flow cytometry is gradually being developed for the quantification of micro- and nano-plastics. A flow cytometer can count particles and analyze their size, shape, and complexity through scatter signals and fluorescence signals. These signals are collected by specific sensors and automatically analyzed, and visually presented in the form of data and charts. The main advantages of flow cytometry are: 1) short detection time, fast speed, and simple operation; 2) high spatial resolution and good recognition effect for micro- and nano-particles; 3) no need for visual interpretation and easy to achieve detection standardization.

[0005] Chinese Patent No. CN 117740653 A discloses a method for detecting sub-micron microplastics in plants based on flow cytometry, which includes the following steps: Step 1) Acid digestion and alkali neutralization treatments are carried out on plant samples, and the neutralized liquid is filtered to obtain an extract; Step 2) The extract obtained in Step 1) is filtered again; Step 3) The particles on the filter membrane in Step 2) are collected and fixed in volume, a surfactant is added for dispersion and a fluorescent dye is added for staining to obtain a stained sample; Step 4) The stained sample obtained in Step 3) is detected by a flow cytometer, and the number of MPs is recorded.

[0006] However, for environmental water samples, how to exclude the interference of inorganic and other organic impurity particles and how to achieve accurate zoning and quantification of microplastics in different particle size ranges are the keys to the development of flow cytometry detection technology. Summary of the Invention

[0007] The present invention provides a method for quantitatively detecting microplastics in water based on Nile red staining-cell-like counting, which can effectively exclude the interference of other impurity particles on the detection results and automatically count small particle size microplastic particles with a size of 1-100 μm by zone.

[0008] The technical solution of the present invention is as follows:

[0009] A method for quantitatively detecting microplastics in water based on Nile red staining-cell-like counting includes the following steps:

[0010] (1) A standard water sample is prepared using microplastics with different known particle size ranges, and the standard water sample is filtered by suction using a filter membrane. After the suction filtration is completed, the particles intercepted on the filter membrane are eluted; a digestion solution is added to the eluate for digestion treatment; then a fluorescent dye is used to stain the digested water sample; the stained water sample is detected by a flow cytometer to obtain the FSC channel signal range of microplastics in different particle size ranges;

[0011] (2) The water sample to be tested is filtered by suction using a filter membrane. After the suction filtration is completed, the particles intercepted on the filter membrane are eluted; a digestion solution is added to the eluate for digestion treatment; then a fluorescent dye is used to stain the digested water sample; the stained water sample is detected by a flow cytometer, and according to the FSC channel signal range of microplastics in different particle size ranges obtained in step (1), microplastics in different particle size ranges are counted respectively.

[0012] The FSC channel signal in the flow cytometer characterizes the size of particles. For particles with a certain size, their FSC signal values are certain. In step (1) of the present invention, first, according to the detection signal values of microplastics within a known particle size range, the FSC signal range of microplastics with different particle sizes in the flow cytometer is obtained, serving as the basis for partitioning the particle sizes of the subsequent water samples to be measured; in step (2), the microplastic counts within different FSC signal ranges are respectively counted, thereby achieving precise partitioning and quantification of microplastics within different particle size ranges.

[0013] Microplastics in different particle size ranges are respectively microplastics with 1 μm ≤ average particle size < 5 μm, 5 μm ≤ average particle size < 10 μm, 10 μm ≤ average particle size < 20 μm, and 20 μm ≤ average particle size ≤ 100 μm.

[0014] The present invention can precisely partition and quantify microplastics within different particle size ranges, enabling a more accurate understanding of the number of microplastics within each particle size range and clarifying their risk hazards, which is of great significance. This is because: firstly, the particle size distribution of microplastics usually follows a negative exponential distribution, and the number of particles will increase exponentially with the decrease in particle size. Therefore, it is necessary to partition particles within different particle size ranges for convenient statistics; secondly, particles within different particle size ranges have different environmental stabilities. For example, particles with a size of 1 - 5 μm have a larger specific surface area and are more likely to become attachment and transmission media for microorganisms, viruses, etc. in the environment; thirdly, particles within different particle size ranges have different health risks. Particles of different sizes have different ingestion pathways and transfer efficiencies when ingested by organisms. Microplastic particles with a size < 10 μm can enter the subcutaneous area through epithelial cells via endocytosis and reach the blood through the lymphatic circulation transported by dendritic cells, while microplastics with a size of 5 - 110 μm mainly enter the circulatory system through the paracellular diffusion effect by inducing inflammation.

[0015] The filter membrane described includes one of a mixed fiber membrane, a polycarbonate membrane, a nitrocellulose membrane, a polytetrafluoroethylene membrane, and a glass fiber membrane.

[0016] The elution methods include flushing elution and ultrasonic elution. The specific steps of flushing elution are: rinsing the filter membrane with 30 ml of ultrapure water, trying to ensure that the entire area is rinsed, and transferring the rinsing solution to a centrifuge tube; the specific steps of ultrasonic elution are: placing the filter membrane in a centrifuge tube, adding 30 ml of ultrapure water, and ultrasonicating in an ultrasonic cleaner for 15 min at a frequency of 40 kHz.

[0017] The digestion solution described is a 30 - 35% H 2 O 2 solution; the digestion treatment is heating and digesting at 50 - 80°C.

[0018] The digestion treatment can effectively eliminate the interference of other organic impurity particles on the detection results.

[0019] The staining procedure includes: fully homogenizing the digested sample, taking the sample into a flow tube, adding Nile Red dye for staining, and immediately performing on-machine detection after staining.

[0020] Furthermore, the digested sample is oscillated by a vortex mixer for 0.5 - 5 min to ensure that the suspension is fully homogenized.

[0021] Dyes with different concentrations have different staining effects on microplastics.

[0022] Preferably, the concentration of Nile Red dye in the sample is 0.1 - 100 μg / mL.

[0023] More preferably, the concentration of Nile Red dye in the sample is 0.5 - 5 μg / mL. At this concentration, the fluorescence signal of microplastic particles is strong enough and dye aggregates are not easily formed.

[0024] More preferably, the concentration of Nile Red dye in the sample is 0.5 - 2 μg / mL.

[0025] Furthermore, the staining time is 5 - 60 min; more preferably 20 - 40 min. At this staining time, the fluorescence signal of microplastic particles is strong enough and dye quenching does not easily occur.

[0026] Before performing flow cytometer detection, the flow cytometer is run at high speed with ultrapure water for 1 - 5 min to ensure that there are no other interferences in the instrument channels.

[0027] When performing flow cytometer detection, the sample loading flow rate is 20 - 40 μL / min, and the scattered signals are received by the forward scatter channel FSC and the side scatter channel SSC, and the fluorescence signal is received by the fluorescence channel.

[0028] Furthermore, the fluorescence channels are FITC (excitation wavelength: 488 nm, emission wavelength: 525 nm), PE (excitation wavelength: 488 nm, emission wavelength: 585 nm), PC5.5 (excitation wavelength: 488 nm, emission wavelength: 690 nm), or APC (excitation wavelength: 638 nm, emission wavelength: 660 nm).

[0029] All four channels can effectively distinguish inorganic particles from microplastic particles with a size of ≥10 μm. For microplastic particles with a size of <10 μm, the fluorescence signal in the FITC channel is weak and there is no obvious difference from the inorganic particle signal; although the PE and APC channels can effectively distinguish microplastic particles with a size of more than 5 μm, for particles with a size of <5 μm, part of their signal peaks overlap with the inorganic particles, resulting in false positives in the counting results; while in the PC5.5 channel, even microplastic particles with a size of 1 μm can be well distinguished from inorganic particles, and the fluorescence signal multiple is 102 around, and there is also a good gradient distribution law among microplastic particles of each particle size.

[0030] Further preferably, the fluorescence channel is PC5.5 (excitation wavelength: 488 nm, emission wavelength: 690 nm).

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] (1) By using the specific staining of Nile red dye for microplastics, the detection method of the present invention can effectively identify microplastics through the fluorescence characteristics of microplastics, and effectively distinguish microplastics from other impurity particles through the gating strategy of the flow cytometer, thereby effectively eliminating the interference of other impurity particles on the detection results;

[0033] (2) The detection method of the present invention provides a lower detection particle size range of 1-100 μm, making up for the shortcoming of the existing methods that are difficult to count small particles, especially particles with a particle size of 1-5 μm that cannot be accurately counted by infrared spectroscopy and Raman spectroscopy;

[0034] (3) The detection method of the present invention simplifies the traditional quantification steps, reduces the subsequent data processing time, and can detect 10 5 particles within 1 minute, realizing rapid, automatic and zonal quantification of microplastics. Description of the Drawings

[0035] Figure 1 It is the flow cytometry zonal map of the polystyrene standard microplastics in Example 1 of the present invention;

[0036] Figure 2 It is the linear relationship diagram between the microplastic mass concentration and the flow cytometry counting result in Example 1 of the present invention;

[0037] Figure 3 It is the signal division map of inorganic particles and microplastic particles in the flow cytometer in Example 2 of the present invention;

[0038] Figure 4 It is the FSC-SSC contour map of different dye concentrations (1 mg / L and 10 mg / L) in Example 3 of the present invention;

[0039] Figure 5 It is the signal distribution of microplastic particles and inorganic particles (N) in four channels (FITC, PE, PC5.5, APC) in Example 4 of the present invention;

[0040] Figure 6 It is the flow cytometry zonal map of the actual tap water sample in Example 6 of the present invention. Detailed Embodiments

[0041] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be noted that the following embodiments are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0042] Example 1

[0043] This example discloses a method for quantitatively detecting microplastics in water based on Nile red staining - flow cytometry analysis, including the following steps:

[0044] Prepare 1 L of samples with different mass concentrations using polystyrene microplastics with particle sizes of 1, 5, 10, and 20 μm. Perform suction filtration using a mixed fiber membrane with a pore size of 0.22 μm. Rinse the front side of the filter membrane with 30 ml of ultrapure water, and collect the rinsing solution into a centrifuge tube. Add 3 ml of 30% H 2 O 2 solution, heat and digest in a water bath at 50°C for 24 h, then shake and mix evenly for 1 min. Take 1 ml of the sample into a flow tube, vertically add 100 μL of Nile red dye with a concentration of 10 mg / L at the tube mouth, and stain in the dark for 30 min. Perform flow cytometry detection at an excitation wavelength of 488 nm and an emission wavelength of 690 nm. The sample flow rate is 30 μL / min, and the recording time is 60 seconds.

[0045] As Figure 1 shown, different particle intervals are divided according to the signal values of the fluorescence channel and the scattering channel.

[0046] The FSC channel signal in the flow cytometer characterizes the size of the particles. For particles with a certain size, their FSC signal values are certain. Therefore, according to the detection signal values of polystyrene microplastics with particle sizes of 1, 5, 10, and 20 μm, the signal ranges of these particles with different particle sizes can be recorded in the flow cytometer, serving as the basis for partitioning subsequent actual water samples.

[0047] Count the polystyrene microplastics, and analyze the linear relationship between the mass concentration of microplastics and the flow cytometry counting results. The average recovery rate of polystyrene microplastics is 87.9%.

[0048] As Figure 2As shown, there is a good linear relationship between the microplastic mass concentration and the flow cytometry counting results, verifying the accuracy of the counting results. The linear relationship between the 1-μm microplastic mass concentration and the flow cytometry counting results is y = 106.2x - 33.841, with R2 = 0.9913; the linear relationship between the 5-μm microplastic mass concentration and the flow cytometry counting results is y = 9.649x + 2.4543, with R2 = 0.9984; the linear relationship between the 10-μm microplastic mass concentration and the flow cytometry counting results is y = 2.0518x + 0.0403, with R2 = 0.9921; the linear relationship between the 20-μm microplastic mass concentration and the flow cytometry counting results is y = 0.2051x - 0.0414, with R2 = 0.9908; where y is the flow cytometry counting result (number / μL) and x is the mass concentration (mg / L).

[0049] Example 2

[0050] This example discloses a method for quantitatively detecting microplastics in water based on Nile red staining - flow cytometry analysis under the interference of inorganic particles, comprising the following steps:

[0051] Mix 5-μm silica inorganic particles with a concentration of 5 mg / L and 5-μm polystyrene microplastics with a concentration of 15 mg / L to prepare 1 L of solution. Use a mixed fiber membrane with a pore size of 0.22 μm for suction filtration. Rinse the front side of the filter membrane with 30 ml of ultrapure water, and collect the rinsing solution into a centrifuge tube. Add 3 ml of 30% H 2 O 2 solution to the above centrifuge tube, heat and digest at 50 °C in a water bath for 24 h, then shake and mix evenly for 1 min. Take 1 ml of the sample into a flow cytometry tube, vertically add 100 μL of Nile red dye with a concentration of 10 mg / L at the tube mouth, and stain in the dark for 30 min. Perform flow cytometry detection at an excitation wavelength of 488 nm and an emission wavelength of 690 nm. The sample flow rate is 30 μL / min, and the recording time is 60 seconds.

[0052] As Figure 3 shown, inorganic particles and microplastic particles can be distinguished in the flow cytometer. The quantitative result of the microplastic sample without adding inorganic particles is 155.17 number / μL, and the quantitative result of the microplastics in the mixed sample with added inorganic particles is 153.90 number / μL. The change in the two quantitative results is only 0.8%, indicating that the detection method of the present invention can well distinguish inorganic particles from microplastic particles, and the microplastic quantitative result is hardly affected by inorganic particles.

[0053] Example 3

[0054] This example discloses a method for quantitatively detecting microplastics in water based on Nile red staining - flow cytometry analysis, comprising the following steps:

[0055] Prepare 1 L of samples with different mass concentrations using polystyrene microplastics with particle sizes of 1, 5, 10, and 20 μm. Perform suction filtration using a mixed fiber membrane with a pore size of 0.22 μm. Rinse the front side of the filter membrane with 30 ml of ultrapure water, collect the rinsing solution into a centrifuge tube, add 3 ml of 30% H 2 O 2 solution, heat and digest in a water bath at 50 °C for 24 h, then shake and mix evenly for 1 min. Take 1 ml of the sample into a flow tube, vertically add 100 μL of Nile red dyes with concentrations of 10 mg / L and 100 mg / L respectively at the tube mouth, and stain in the dark for 30 min. Perform flow cytometry detection at an excitation wavelength of 488 nm and an emission wavelength of 690 nm, with a sample flow rate of 30 μL / min and a recording time of 60 seconds.

[0056] As Figure 4 shown, a dye concentration of 1 mg / L can achieve a good balance among the fluorescence signal intensity, the formation of dye aggregates, and the quenching of the dye. Particle aggregation occurred in the test tube with a concentration of 10 mg / L, especially for particles >20 μm. Among them, the particles of 1 - 5 μm and 5 - 10 μm decreased by about 57% compared to the test tube with 1 mg / L, while the particles of 10 - 20 μm and 20 - 100 μm increased by about 3 times and 9 times respectively.

[0057] Example 4

[0058] This example discloses a method for quantitatively detecting microplastics in actual water bodies based on Nile red staining - flow cytometry analysis, including the following steps:

[0059] Prepare 1 L of samples with different mass concentrations using polystyrene microplastics with particle sizes of 1, 5, 10, and 20 μm. Perform suction filtration using a mixed fiber membrane with a pore size of 0.22 μm. Rinse the front side of the filter membrane with 30 ml of ultrapure water, collect the rinsing solution into a centrifuge tube, add 3 ml of 30% H 2 O 2 solution, heat and digest in a water bath at 50 °C for 24 h, then shake and mix evenly for 1 min. Take 1 ml of the sample into a flow tube, vertically add 100 μL of Nile red dye with a concentration of 10 mg / L at the tube mouth, and stain in the dark for 30 min. Perform flow cytometry detection at an excitation wavelength of 488 nm, emission wavelengths of 525 nm, 585 nm, 690 nm, and an excitation wavelength of 638 nm, emission wavelength of 660 nm, with a sample flow rate of 30 μL / min and a recording time of 60 seconds.

[0060] As Figure 5As shown, all four channels can effectively distinguish inorganic particles from microplastic particles with a size of ≥10 μm. For microplastic particles with a size of <10 μm, the fluorescence signal in the FITC channel is weak and there is no obvious difference from the inorganic particle signal; although the PE and APC channels can effectively distinguish microplastic particles with a size of more than 5 μm, for particles with a size of <5 μm, a part of their signal peaks overlaps with inorganic particles, resulting in false positives in the counting results; while in the PC5.5 channel, it is found that even microplastic particles with a size of 1 μm can be well distinguished from inorganic particles, and the fluorescence signal multiple is about 10 2 or so, and there is also a good gradient distribution law among microplastic particles of different particle sizes.

[0061] Example 5

[0062] This example discloses a method for quantitatively detecting microplastics in water based on Nile red staining - flow cytometry analysis under the interference of organic particles, including the following steps:

[0063] Equal amounts of polystyrene microplastics were added to 2 bottles of 1L tap water each containing organic particles, and vacuum filtration was carried out using a mixed fiber membrane with a pore size of 0.22 μm. The front side of the filter membrane was rinsed with 30 ml of ultrapure water, and the rinsing solution was collected into a centrifuge tube. 3 ml of 30% H 2 O 2 solution was added to one of the above centrifuge tubes, and it was heated and digested in a water bath at 50 °C for 24 h; the other one was not treated with digestion. Subsequently, they were respectively shaken and mixed evenly for 1 min, 1 ml of the sample was taken into a flow tube, and 100 μL of Nile red dye with a concentration of 10 mg / L was added vertically to the tube opening, and stained in the dark for 30 min. Flow cytometry detection was carried out at an excitation wavelength of 488 nm and an emission wavelength of 690 nm, the sample flow rate was 30 μL / min, and the recording time was 60 seconds. The results showed that compared with the undigested tap water sample, the number of particles after digestion decreased by 16%, indicating that the digestion method of treating with 30% H 2 O 2 at 50 °C for 24 h can remove organic particles in the actual water sample.

[0064] Example 6

[0065] This example discloses a method for quantitatively detecting microplastics in actual water bodies based on Nile red staining - flow cytometry analysis, including the following steps:

[0066] Take 1L of tap water, carry out vacuum filtration using a mixed fiber membrane with a pore size of 0.22 μm, rinse the front side of the filter membrane with 30 ml of ultrapure water, collect the rinsing solution into a centrifuge tube, and add 3 ml of 30% H 2 O 2The solution was heated and digested in a water bath at 50 °C for 24 h, then shaken and mixed evenly for 1 min. 1 ml of the sample was taken into a flow tube, and 100 μL of Nile red dye with a concentration of 10 mg / L was added vertically to the tube mouth, and stained in the dark for 30 min. Flow cytometry detection was carried out at an excitation wavelength of 488 nm and an emission wavelength of 690 nm. The sample flow rate was 30 μL / min, and the recording time was 60 seconds.

[0067] As Figure 6 shown, according to the different signal values of particles with different particle sizes, automatic counting was carried out by a flow cytometer within a certain range. The number concentration of microplastic particles with a size of 1-100 μm in this tap water sample was 167,750 particles / L, among which the number concentration of microplastic particles with a size of 1-5 μm was 130,597 particles / L, the number concentration of microplastic particles with a size of 5-10 μm was 33,028 particles / L, the number concentration of microplastic particles with a size of 10-20 μm was 2,805 particles / L, and the number concentration of microplastic particles with a size of 20-100 μm was 1,320 particles / L.

[0068] The above-described embodiments have detailed the technical solutions and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the present invention. Any modifications, supplements, equivalent replacements, etc. made within the principle scope of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for quantitatively detecting microplastics in water based on Nile red staining-like cell counting, characterized in that: The following steps are involved: (1) Prepare a standard water sample containing microplastics of different particle size ranges and known particle sizes, use a filter membrane to filter the standard water sample, and after the filtration is completed, elute the particles trapped on the filter membrane; add a digestion solution to the eluent for digestion treatment; then use a fluorescent dye to stain the digested water sample; and perform flow cytometry on the stained water sample to obtain the FSC channel signal interval of microplastics of different particle size ranges; (2) Using a filter membrane to filter the water sample to be tested, after the filtration is completed, the particles retained on the filter membrane are eluted; a digestion solution is added to the elution solution for digestion treatment; the digested water sample is then stained with a fluorescent dye; the stained water sample is tested by flow cytometry, and the microplastics in different particle size ranges are counted according to the FSC channel signal intervals of the microplastics in different particle size ranges obtained in step (1); Microplastics in different particle size ranges are microplastics with an average particle size of 1 μm ≤ < 5 μm, an average particle size of 5 μm ≤ < 10 μm, an average particle size of 10 μm ≤ < 20 μm, and an average particle size of 20 μm ≤ ≤ 100 μm. The digestion solution is a 30-35% H2O2 solution; the digestion treatment is heating digestion at 50-80°C; When performing flow cytometry detection, the sample flow rate is 20-40 μL / min, the radial scattering channel FSC and the side scattering channel SSC receive scattering signals, and the fluorescence channel receives fluorescence signals; the fluorescence channel is PC5.5, the excitation wavelength is 488nm, and the emission wavelength is 690nm.

2. The method for quantitatively detecting microplastics in water based on Nile red staining-like cell counting according to claim 1 is characterized in that: Elution methods include flushing elution and ultrasonic elution; The specific steps of flushing and elution are as follows: flush the filter membrane with 30 ml of ultrapure water, try to flush the entire area, and pour the flushing solution into a centrifuge tube; The specific steps of ultrasonic elution are as follows: place the filter membrane in a centrifuge tube, add 30 ml of ultrapure water, and ultrasonicate in an ultrasonic cleaning machine for 15 min at a frequency of 40 kHz.

3. The method for quantitatively detecting microplastics in water based on Nile red staining-like cell counting according to claim 1 is characterized in that: The staining steps include: fully homogenizing the digested sample, taking the sample into a flow tube, adding Nile red dye for staining, and immediately testing on a machine after staining.

4. The method for quantitatively detecting microplastics in water based on Nile red staining-like cell counting according to claim 3 is characterized in that: The concentration of Nile red dye in the sample was 0.1-100 μg / mL.

5. The method for quantitatively detecting microplastics in water based on Nile red staining-like cell counting according to claim 4 is characterized in that: The concentration of Nile red dye in the samples was 0.5-5 μg / mL.

6. The method for quantitatively detecting microplastics in water based on Nile red staining-like cell counting according to claim 3 is characterized in that: The staining time is 5-60min.

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