Integrated analysis method for enrichment and Raman detection of micro-plastics in biological tissues

Abstract

The invention discloses an integrated analysis method for enrichment and Raman detection of micro-plastics in biological tissues, and belongs to the technical field of enrichment extraction and detection of micro-plastics. The method comprises the following steps: pre-treating biological tissues through a digestion solution, and filtering a tissue sample digestion solution through a silver filter membrane; dyeing by using a Nile red standard stock solution, and observing and counting the micro-plastic particles by using a fluorescence microscope; all Raman spectrograms of suspected micro-plastic particles enriched on the silver filter membrane are paired with a standard substance reference Raman database on the basis of KnowItAll software. The method provided by the invention has high efficiency and high recovery rate, can accurately detect the MPs component in the tissue in a short time, so that the detection precision is improved, the cost is lower, and the method can be used for a long time, so that the detection cost is reduced; the effective dyeing of the MPs can be realized, so that the fluorescence intensity characterization of the dyed MPs can be stably and accurately observed.

Description

Technical Field

[0001] The present invention belongs to the technical field of microplastic enrichment, extraction and detection, and specifically relates to an integrated analysis method for microplastic enrichment and Raman detection in biological tissues. Background Art

[0002] In recent years, microplastic (MPs) pollution has become a global concern. MPs are plastic particles with a diameter of less than 5 mm, including primary and secondary MPs. These MPs are widely present in the environment, and their traces can be detected from the ocean to freshwater, from soil to air. Due to their small size, MPs are easily ingested by organisms and enter the food chain, posing a potential threat to ecosystems and human health.

[0003] The accumulation of microplastics (MPs) in biological tissues has attracted increasing attention. Studies have shown that MPs can enter the body through a variety of pathways, such as drinking water, food, and air inhalation. Microplastics that enter the body not only trigger inflammatory responses but also potentially exacerbate health risks by adsorbing toxic chemicals on their surfaces. Therefore, in-depth research on the accumulation of MPs in biological tissues and their detection methods is of great significance.

[0004] Currently, methods for detecting MPs in biological tissues primarily include microscopy, pyrolysis gas chromatography-mass spectrometry (Py-GC / MS), and Fourier transform infrared spectroscopy (FTIR). While these methods have played an important role in MPs detection, they also have limitations. For example, microscopy relies heavily on operator experience and is difficult to achieve quantitative analysis; Py-GC / MS requires complex sample pretreatment and is expensive; and FTIR can be susceptible to matrix interference when detecting MPs, resulting in reduced sensitivity.

[0005] Raman spectroscopy, a nondestructive testing technique, has recently demonstrated great potential for MPs detection. It can provide molecular fingerprint information and offers advantages such as high sensitivity, high resolution, and ease of use. However, traditional Raman spectroscopy methods still face technical challenges in detecting MPs in biological tissues, such as weak signals, strong background interference, and complex sample preparation. Summary of the Invention

[0006] To address the above issues, the present invention provides a method for detecting MPs in biological tissues, accurately detecting the composition of MPs in biological tissues. The method of the present invention is efficient, economical, minimally interfering, and highly accurate, providing a new pretreatment technology for the determination of MPs in biological tissue samples.

[0007] The first object of the present invention is to provide a method for detecting MPs in biological tissues, comprising the following steps:

[0008] (1) digesting the biological tissue with a digestion solution to obtain a digestion solution of the digested tissue sample, and then diluting the solution; the digestion solution includes an alkaline solution and an H2O2 solution;

[0009] (2) filtering the diluted tissue sample digestion solution through a filter membrane using a glass vacuum filtration system, washing, drying, and obtaining a filter membrane with tissue sample residue;

[0010] (3) staining the filter membrane with tissue sample residues from step (2) using a Nile red standard stock solution, and observing and counting the microplastic particles using a fluorescence microscope;

[0011] (4) Obtain all Raman spectra of suspected microplastic particles enriched on the filter membrane in step (2), match the obtained Raman spectra with the standard reference Raman database based on the KnowItAll software, and screen the spectra with a similarity of more than 70% with the standard reference spectrum.

[0012] In one embodiment of the present invention, in step (1), the biological tissue is human tissue or animal tissue; the human tissue is uterine fibroid tissue, and the animal tissue is fresh meat.

[0013] In one embodiment of the present invention, in step (1), the biological tissue is firstly digested with an alkaline solution and then digested with an H2O2 solution.

[0014] In one embodiment of the present invention, in step (1), the alkaline digestion time is 12-72 hours, more preferably 48 hours, the alkaline solution is KOH solution, the mass concentration of the KOH solution is 8-12%, and the alkaline digestion temperature is 50°C.

[0015] In one embodiment of the present invention, in step (1), the mass volume ratio of the biological tissue to the alkaline solution is 1-1.1:30, g / mL.

[0016] In one embodiment of the present invention, in step (1), the digestion time of the H2O2 solution is 24 hours, the mass concentration of the H2O2 solution is 25-35%, and the digestion temperature is 28°C.

[0017] In one embodiment of the present invention, in step (1), the mass volume ratio of the biological tissue to the acid solution is 1-1.1:5, g / mL.

[0018] In one embodiment of the present invention, in step (1), the tissue sample digestion solution is diluted with ultrapure water, and the mass volume ratio of biological tissue to ultrapure water is 1-1.1:600, g / mL.

[0019] In one embodiment of the present invention, in step (2), the filter membrane is a silver filter membrane.

[0020] In one embodiment of the present invention, in step (3), one or more of methanol, chloroform, acetone, and n-hexane are selected to prepare a Nile Red (NR) standard stock solution, preferably acetone; the concentration of the Nile Red standard stock solution is 1 mg / mL.

[0021] In one embodiment of the present invention, in step (3), the NR standard stock solution is diluted to 20 μg / mL using a mixture of acetone and ethanol in a volume ratio of 1:1, and then the filter membrane with the tissue sample residue is stained.

[0022] In one embodiment of the present invention, in step (3), dyeing is performed in an oven or a water bath, more preferably in a water bath; the dyeing temperature is 60° C., and the dyeing time is 30 min.

[0023] In one embodiment of the present invention, in step (3), the wavelength selection for fluorescence microscopy is an excitation wavelength of 450-490 nm and an emission wavelength of 515-565 nm.

[0024] In one embodiment of the present invention, in step (4), the method of obtaining all spectra of the Raman spectrum of the suspected MPs particles enriched on the filter membrane includes: using a micro Raman spectrometer using a 50× objective lens to morphologically characterize the observed suspected MPs particles, and then using the Raman spectrometer to obtain the spectrum of the suspected MPs polymer.

[0025] In one embodiment of the present invention, in step (4), the laser wavelength of the micro-Raman spectroscopy is 532 nm, and the spectral scanning range is 200-4000 cm -1 .

[0026] In one embodiment of the present invention, in step (4), the database includes the KnowItAll software built-in spectral library, SLoPP and SLoPP-E Raman spectral libraries, and the RDWP MP Raman database developed for studying MPs.

[0027] Beneficial effects of the present invention:

[0028] (1) The biological tissue sample pretreatment method established in the present invention not only completely digests the organic components in the tissue sample, but also has high efficiency and high recovery rate. The total digestion time is 72 hours, and the recovery rates of 1μm and 10μm polyethylene are 89.48%±4.47% and 96.27%±2.48%, respectively.

[0029] (2) The present invention uses micro-Raman spectroscopy to obtain all spectra of the Raman spectra of the suspected microplastic particles enriched on the filter membrane, and combines KnowItAll software and database to analyze the obtained spectra. It can accurately detect the MPs components in the tissue in a short time, thereby improving the detection accuracy, and is low in cost and can be used for a long time, thereby reducing the detection cost.

[0030] (3) The present invention studied a mixed dye consisting of acetone and ethanol in a volume ratio of 1:1 and a concentration of 20 μg / mL, which can effectively stain MPs in a 60°C water bath. This allows the fluorescence intensity of the stained MPs to be stably and accurately observed.

[0031] (4) The present invention provides an integrated analytical method suitable for the enrichment and Raman detection of microplastics in biological tissues, which has the advantages of simple operation, sensitive detection, and strong applicability, and can efficiently realize the separation and identification of microplastics in complex biological tissues. The detection of the composition and distribution of microplastics in human tissues by this method not only helps to reveal the migration and accumulation mechanism of microplastics in the body, but also provides a scientific basis for evaluating their potential toxicity and health risks, and promotes in-depth research on the relationship between environmental exposure and human health. At the same time, this method is suitable for the detection of microplastics in animal tissues, especially in food-packaged meat products. It can be used to analyze the release of microplastics in packaging materials and their residual levels in meat tissues, thereby providing technical support for food safety risk assessment and packaging material safety monitoring. This method has broad application prospects and important practical significance in the fields of public health, food safety and environmental health. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the process of MPs enrichment, extraction and Raman analysis in biological tissues in Example 1.

[0033] Figure 2 This is the turbidity standard curve in Example 2.

[0034] Figure 3 It is the turbidity value of the digestive fluid of tissue at different digestion times in Example 2.

[0035] Figure 4 These are the recovery values of the spiked polyethylene (PE) MPs with sizes of 1 μm and 10 μm in Example 2.

[0036] Figure 5 Fluorescence imaging of the optimized NR staining conditions in Example 3; A. methanol, B. n-hexane, C. CHCl3, D. acetone, E. oven, F. water bath.

[0037] Figure 6 This is the fluorescence imaging image of MPs observed in biological tissue in Example 4.

[0038] Figure 7 This is the SEM image of representative MPs in biological tissue in Example 5.

[0039] Figure 8 These are Raman spectra of representative MPs in biological tissues in Examples 6 and 7. DETAILED DESCRIPTION

[0040] The following describes preferred embodiments of the present invention. It should be understood that these embodiments are intended to better illustrate the present invention and are not intended to limit the present invention. Reagent materials, such as polyethylene MPs standard, were purchased from Dongguan Wensheng Plastics Raw Materials Co., Ltd.; NR (≥95%), acetone, n-hexane, chloroform, H2O2, and ethanol were supplied by Sinopharm Chemical Reagent Co., Ltd.

[0041] The uterine fibroid tissue samples used in the examples were obtained from Wuxi Maternal and Child Health Hospital and the Affiliated Hospital of Jiangnan University. The use of these samples was approved by the Medical Ethics Committee of the Affiliated Hospital of Jiangnan University (LS2021019). All participants signed informed consent forms.

[0042] Example 1

[0043] An integrated analytical method for microplastic enrichment and Raman detection in biological tissues, comprising the following steps:

[0044] (1) Establishment of tissue sample pretreatment method:

[0045] Accurately weigh 1 g of human uterine fibroid tissue sample, rinse with pre-filtered ultrapure water, and transfer to a 50 mL glass Erlenmeyer flask. Then, add 30 mL of 10% KOH solution to the flask, and seal it with a glass stopper. The sample is then placed in a 50°C incubator with constant agitation at 120 rpm for 48 hours. After digestion, cool the sample to room temperature, add 5 mL of 30% H₂O₂ solution, and digest again in a shaker at 28°C at 120 rpm for 24 hours.

[0046] The digestion solution was diluted with 600 mL of ultrapure water and divided into ten equal parts. The diluted sample suspension was then filtered through a silver filter membrane (pore size: 0.45 μm, diameter: 25 mm) using a glass vacuum filtration system. After filtration, the glass conical flask and vacuum filtration apparatus were washed with ultrapure water (200 mL), and the washing solution was filtered again. The silver filter membrane with the filter residue was rinsed with 50 mL of ultrapure water, then transferred to a glass culture dish (diameter = 35 mm) and dried in an oven at 60° C. for 5 h. The dried silver filter membrane was stored at room temperature for further analysis.

[0047] (2) Raman detection method of MPs:

[0048] The samples were analyzed by micro-Raman spectroscopy. The morphology of the suspected MPs particles was characterized using a 50× objective lens, and the spectrum of the suspected MPs polymer was obtained using a Raman spectrometer (laser 532, slit 600, hole 800, wavelength selection 200-4000cm -1 All detected Raman spectra were compared with those reported in the literature, including the spectral library of KnowItAll software (WileyScience Solutions, Hoboken, NJ, USA) and the freely available SLOPP / SLOPPe libraries of known microplastics. A Raman spectral threshold of ≥70% indicated the presence of suspected plastic polymers in the sample.

[0049] The enrichment, extraction and Raman analysis process of MPs in biological tissues is as follows: Figure 1 shown.

[0050] Example 2

[0051] Optimization of time for digestion of tissue samples:

[0052] (1) Preparation of turbidity standard curve:

[0053] Turbidity standard solution: Pipette 5 mL of hydrazine sulfate solution and 5 mL of 4-methyltetramine solution into a 100 mL volumetric flask and mix thoroughly. Allow to react at 25 ± 3°C for 24 hours. After cooling, dilute to the mark with water and mix thoroughly.

[0054] To draw a standard curve: aspirate 0, 0.50, 1.25, 2.50, 5.00, 10.00 and 12.50 mL of turbidity standard solution into a 50 mL colorimetric tube, and add turbidity-free water to the mark. After shaking, the standard system with turbidity of 0, 4, 10, 20, 40, 80, and 100 is obtained at a wavelength of 680 nm. Use a 30 mm colorimetric dish to measure the absorbance and draw a standard curve. Figure 2 As shown, hydrazine sulfate and 4-methyltetramine were used to establish a turbidity standard curve R 2 =0.9986.

[0055] (2) Optimization of time for digestion of tissue samples:

[0056] The effects of digestion times of 12h, 24h, 48h and 72h on the digestion efficiency of tissue samples were studied.

[0057] Accurately weigh 1g of human uterine fibroid tissue sample, rinse with pre-filtered ultrapure water, and transfer to a 50mL glass Erlenmeyer flask. Add 30mL of 10% KOH solution to the flask, and seal it with a glass stopper. The sample was then placed in a 50°C incubator with continuous agitation at 120 rpm for 12, 24, 48, and 72 hours. Each digestion time was repeated three times.

[0058] like Figure 3 As shown in Figure A, the turbidity values after 48 and 72 hours of digestion were 233.80 ± 6.22 and 230.07 ± 4.98 NTU, respectively, with no significant difference (p>0.05). The turbidity value at 24 hours was 283.59 ± 6.22 NTU, significantly higher than the values at 48 and 72 hours (p<0.05). Therefore, after 24 hours of digestion, the tissue was not completely digested. After 48 hours, the tissue was almost completely digested, with no significant changes thereafter, which is consistent with visual observation. Therefore, 48 hours was selected as the optimal digestion time for the first digestion.

[0059] Because biological samples contain a large amount of organic matter, even if 10% KOH is used to digest tissue samples, the organic matter cannot be completely digested. Therefore, 30% H2O2 is used for secondary digestion to digest the residual organic matter and remove lipid and protein interference. Figure 3 As shown in Figure B, after 24 hours of digestion with 30% H₂O₂, the turbidity of the digestate was 109.20 ± 3.73 NTU, significantly lower than that at 48 hours (p < 0.05). The tissue can be considered completely digested. Therefore, 72 hours was selected as the optimal digestion time for both digestions.

[0060] Test of spike recovery:

[0061] 10 mg of polyethylene MPs standard material (sizes of 1 μm and 10 μm) was added to 1 g of human uterine fibroid tissue sample.

[0062] Repeat step (1) of Example 1 to process the spiked sample.

[0063] Ultrapure water was used for procedural blank analysis. The experiment was repeated three times. After the experiment, the spiked tissue samples were corrected using the data from the unspiked tissue samples, and the recovery of polyethylene MPs was calculated. The recovery (%) was calculated as the ratio of the measured polymer mass (after correction using the blank) to the theoretical spiked mass × 100%.

[0064] like Figure 4When corrected for blank, the recoveries of 1 μm and 10 μm polyethylene MPs were 89.48% ± 4.47% and 96.27% ± 2.48%, respectively. However, the recovery of 10 μm polyethylene MPs was slightly higher than that of 1 μm.

[0065] Example 3

[0066] Optimization of MPs staining method

[0067] (1) Optimization of NR staining method:

[0068] Initial examination of MPs is performed using the Nile red staining protocol, as follows:

[0069] 10 mg of polyethylene MPs standard substance was added to 1 g of human uterine fibroid tissue sample, and step (1) of Example 1 was repeated to treat the spiked sample to obtain a silver filter membrane with filtration residue; the sample was dried in a 40°C oven for 5 h. Methanol, chloroform, acetone, and n-hexane were selected to prepare a 1 mg / mL NR standard stock solution, which was diluted to 20 μg / mL using a v / v mixture of acetone and ethanol at a ratio of 1:1. The dilutions were used for MPs staining. Using a glass syringe, 0.2-0.5 mL of Nile red staining solution was applied to the dried silver membrane filter to stain the microplastics. The microplastics were stained in a 60°C water bath for 30 min.

[0070] (2) Optimization of staining conditions:

[0071] 10 mg of polyethylene MPs standard substance was added to 1 g of human uterine fibroid tissue sample, and step (1) of Example 1 was repeated to treat the spiked sample to obtain a silver filter membrane with filtration residue; the sample was dried in a 40°C oven for 5 h. A 1 mg / mL NR standard stock solution was prepared in acetone, and the NR standard stock solution was diluted to 20 μg / mL using a v / v mixture of acetone and ethanol of 1:1. The dilutions were used for MPs staining. Using a glass syringe, 0.2-0.5 mL of Nile red staining solution was applied to the dried silver membrane filter to stain the microplastics. The microplastics were stained for 30 min in a 60°C water bath and a 60°C oven, respectively.

[0072] (3) Fluorescence microscopy observation:

[0073] The stained and dried silver filter membrane was placed on a glass slide and further examined under a fluorescence microscope (AxioVert A15 / 7KMAT microscope, filter set HE43, AxioCam 503 color, ZEN software, Zeiss, Germany). Emitted fluorescence was used to distinguish particles from the background and count them, while also analyzing particle size and shape. Green fluorescence (excitation wavelength 450-490 nm; emission wavelength 515-565 nm) was used for measurement.

[0074] Figure 5 Fluorescence images of the MPs staining process are shown. Figure 5 As shown in Figures AD, MPs stained with NR dye solution in acetone showed stronger fluorescence intensity than MPs stained with NR dye solution in methanol, chloroform, and n-hexane. The results indicate that NR solution prepared in acetone is the best choice for MPs staining. Figure 5 As shown in Figures EF, the samples stained in a water bath had stronger fluorescence intensity than those stained in an oven. Therefore, heating in a water bath at 60°C for 30 min was finally selected as the heating method for MPs staining.

[0075] Example 4

[0076] 1.05, 1.08, 1.05, 1.06, 1.07, and 1.03 g of human uterine fibroid tissue samples were weighed, respectively, and step (1) of Example 1 was repeated to obtain a silver filter membrane with a filtration residue. A 1 mg / mL NR standard stock solution was prepared in acetone. The NR standard stock solution was diluted to 20 μg / mL using a v / v mixture of acetone and ethanol at a ratio of 1:1. Using a glass syringe, 0.1-0.5 mL of Nile Red staining solution was applied to the dried silver membrane filter and stained in a 60°C water bath for 30 min. Fluorescence microscopy analysis was consistent with step (3) of Example 3.

[0077] like Figure 6 As shown, 1#-6# are the sample numbers, and the weighed masses of the samples are 1.05, 1.08, 1.05, 1.06, 1.07, and 1.03 g, respectively. The results show that the biological tissue samples contain MPs.

[0078] Example 5

[0079] Scanning electron microscopy (SEM) observation of MPs surface morphology includes the following steps:

[0080] The silver filter membrane with the filtration residue obtained in Example 1 was used to select the MPs particles observed by micro-Raman using a 50× objective lens. A scanning electron microscope (SEM; SU8100, resolution: 0.8 nm, Hitachi, Japan) was used to further capture high-quality images of the MPs surface. SEM images of MPs were taken on the substrate at different magnifications (1-4k), focusing on the areas showing significant microstructural changes. Figure 7 SEM images showed that the MPs were spherical, fragmented, and elongated in shape, with a size of less than 20 μm.

[0081] Example 6 Detection of MPs in human tissue

[0082] (1) The biological tissue sample in Example 1 was adjusted to a human uterine fibroid tissue sample. Other procedures remained the same as in Example 1, and pretreatment and Raman detection were performed.

[0083] (2) Accurately weigh 1.06 g, 1.08 g, 1.05 g, 1.09 g, 1.08 g, and 1.04 g of human uterine fibroid tissue samples and rinse the samples with pre-filtered ultrapure water, respectively. The remaining operations are the same as steps (1) and (2) in Example 1.

[0084] (3) The collected Raman spectra were matched with the Raman polymer database, the free SLOPP / SLOPPe plastic database, and the RDWP Raman database in the KnowItAll software (Wiley Science Solutions, Hoboken, NJ, USA). Characteristic peaks were identified, and a similarity of a matching quality index (HQI) ≥ 70 was considered to be the standard for determining the material under this matching degree. Figure 8 As shown, Figure 8 (A)-(F) correspond to human uterine fibroid tissue samples of 1.06g, 1.08g, 1.05g, 1.09g, 1.08g, and 1.04g, respectively. The results show that human tissue contains MPs. The main types of MPs present are polyethylene, polypropylene, polystyrene, polyethylene terephthalate, polyamide, and polyvinyl chloride.

[0085] Example 7 Detection of MPs in Animal Tissues

[0086] Accurately weigh 1.06g, 1.05g, 1.05g, 1.07g, 1.06g, and 1.08g of fresh pork, and rinse the samples with pre-filtered ultrapure water. The remaining operations are the same as in Example 6. Figure 8 As shown, Figure 8(G)-(L) correspond to 1.06g, 1.05g, 1.05g, 1.07g, 1.06g, and 1.08g of pork, respectively. The results indicate that MPs are present in animal tissues. The main types of MPs are polyethylene, polypropylene, polystyrene, polyethylene terephthalate, polyamide, and polyvinyl chloride.

[0087] From the above content, it can be seen that the technical solution provided by the present invention can enrich and extract MPs in biological tissues in a short time, and can realize accurate detection of MPs composition and quality, thereby improving detection accuracy.

[0088] The embodiments provided above are not intended to limit the scope of the present invention, nor are the steps described to limit their execution order. Any obvious improvements to the present invention made by those skilled in the art in combination with existing common knowledge shall fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for detecting microplastics in biological tissues, characterized in that: The steps include: (1) digesting the biological tissue with an alkaline solution and then with H2O2 to obtain a digested tissue sample solution, which is then diluted; (2) filtering the diluted tissue sample digestion solution through a silver filter membrane using a glass vacuum filtration system, washing, drying, and obtaining the silver filter membrane with tissue sample residue; (3) using Nile red standard stock solution to stain the silver filter membrane with tissue sample residues in step (2), and observing and counting microplastic particles using a fluorescence microscope; (4) Obtain all Raman spectra of suspected microplastic particles enriched on the silver filter membrane in step (2), and pair the obtained Raman spectra with the standard reference Raman database based on the KnowItAll software to screen the spectra with a similarity of more than 70% with the standard reference spectrum.

2. The method according to claim 1, characterized in that In step (1), the biological tissue is human tissue or animal tissue.

3. The method according to claim 1, characterized in that In step (1), the alkaline digestion time is 12-72 hours; the alkaline solution is a KOH solution; the mass concentration of the alkaline solution is 8-12%, the alkaline digestion temperature is 50° C.; the mass volume ratio of the biological tissue to the alkaline solution is 1-1.1:30, g / mL.

4. The method according to claim 1, wherein In step (1), the H2O2 digestion time is 24 hours, the mass concentration of H2O2 is 25-35%; the H2O2 digestion temperature is 28°C; and the mass volume ratio of biological tissue to H2O2 is 1-1.1:5, g / mL.

5. The method according to claim 1, wherein In step (1), the tissue sample digestion solution is diluted with ultrapure water, and the mass volume ratio of biological tissue to ultrapure water is 1-1.1:600, g / mL.

6. The method according to claim 1, characterized in that In step (3), one of methanol, chloroform, acetone, and n-hexane is selected to prepare a Nile red standard stock solution; the concentration of the Nile red standard stock solution is 1 mg / mL.

7. The method according to claim 1, characterized in that In step (3), the Nile red standard stock solution was diluted to 20 μg / mL using a mixture of acetone and ethanol in a volume ratio of 1:1, and then the silver filter with tissue sample residue was stained.

8. The method according to claim 1, characterized in that In step (3), dyeing is performed in an oven or a water bath under heating; the dyeing temperature is 60° C., and the dyeing time is 30 min.

9. The method according to claim 1, characterized in that In step (4), the method for obtaining all spectra of the Raman spectrum of the suspected microplastic particles enriched on the silver filter membrane includes: using a micro-Raman spectrometer with a 50× objective lens to morphologically characterize the observed suspected MPs particles, and then using a Raman spectrometer to obtain the spectrum of the suspected MPs polymer.

10. The method according to claim 1, characterized in that In step (4), the database includes the KnowItAll software built-in spectral library, SLoPP and SLoPP-E Raman spectral libraries, and RDWP MP Raman database.

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