Rapid detection method of microplastics in human intestinal tissue based on micro-raman

Abstract

The application discloses a kind of based on micro-Raman to human intestinal tissue microplastics rapid detection method, belong to biological tissue microplastics detection technical field.The alkali solution is used to carry out alkali digestion pretreatment to human intestinal tissue, acid is added to digestion liquid to neutral, and the glass fiber filter membrane is used to filter tissue sample digestion liquid, and the microplastics on filter membrane is washed with ultrapure water;The representative area on filter membrane is imaged using micro-Raman spectrometer mosaic puzzle and panorama, and the Raman spectrum of suspected microplastic particles is collected, and the measured Raman spectrum is compared with database standard spectrum by combining KnowItAll software, to determine its composition and count the number of microplastics on filter membrane.The application can provide technical support for studying the enrichment and migration transformation rule of microplastics in human intestinal tissue and health risk assessment.

Description

Technical Field

[0001] This invention relates to the field of microplastic extraction and detection technology, specifically to a rapid detection method for microplastics in human intestinal tissue based on micro Raman spectroscopy. Background Technology

[0002] In recent years, microplastics (MPs), as a novel global environmental pollutant, have become a research hotspot in the environmental field due to their widespread distribution, recalcitrant degradation, and easy migration. MPs are defined as plastic particles and fragments with a diameter of less than 5 mm. Based on their source, they can be divided into primary and secondary microplastics. They are widely present in environmental media and can enter the human body through various routes, including food, water intake, and air inhalation, easily accumulating in human tissues and organs. The intestine is one of the organs most directly exposed after oral ingestion of microplastics. Larger microplastic particles are usually easily excreted with intestinal contents, while smaller microplastics are more likely to interact with the intestinal mucosal barrier, epithelial cells, and local tissue microenvironment, thereby inducing or aggravating local inflammatory responses, oxidative stress, intestinal barrier function damage, and tumor microenvironment dysbiosis. Therefore, establishing a detection method for MPs in human intestinal tissue is of great significance for revealing the risk assessment of MP exposure in human intestinal tissue.

[0003] Based on their principles, microplastic (MP) detection methods are mainly classified into: spectroscopic methods, mass spectrometry (chromatographic coupled with chromatography), fluorescence staining methods, optical (scattering) methods, and nuclear magnetic resonance spectroscopy. Spectroscopic methods offer advantages in qualitative and quantitative analysis and are the primary methods for microplastic detection, including Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy. FTIR spectroscopy is the most widely used technique in MP research, but due to its diffraction limit, it is difficult to identify particles larger than 10 μm. Microscopic Raman detection systems acquire the Raman spectra of MPs to obtain their "characteristic fingerprint" information. This technology has significant advantages in detecting and identifying smaller-sized plastics (particle size less than 20 μm), possessing high spatial resolution (up to 1 μm) and high sensitivity, and is currently widely used in materials science, life sciences, and other fields. However, the current methods for analyzing microplastics in human intestinal tissue based on micro Raman spectroscopy still have the following limitations in practical applications: (1) There is no unified standard for the detection of microplastics in human intestinal tissue. There are differences in the selection of digestion reagents, reaction time, filter membrane material and pore size, which limits the comparability of detection results; (2) The intestinal tissue matrix is ​​complex and contains a large number of proteins, lipids, cell debris and mucus components. If the digestion treatment is not sufficient, impurities are likely to remain on the surface of the filter membrane, affecting subsequent microscopic observation and Raman spectroscopy identification; if the digestion conditions are too strong or the reaction time is too long, it may affect the morphological integrity or spectral characteristics of some polymer particles; (3) The use of imported filter membranes with higher prices is not conducive to the promotion of clinical tissue sample detection; (4) It is necessary to use fluorescent staining, microscopic screening and other methods to determine the location of suspected microplastic particles. There are many operation steps and the detection cycle is long, which increases the risk of sample transfer, exogenous contamination and particle loss; (5) It is difficult to meet the high sensitivity detection requirements of submicron microplastics in human tissue. Therefore, there is an urgent need to establish a rapid detection method for microplastics in human intestinal tissue based on micro Raman spectroscopy, so as to achieve efficient digestion, low-cost enrichment, simple and rapid detection of complex tissue samples, and identification of submicron-sized microplastic particles. Summary of the Invention

[0004] To address the aforementioned technical challenges, this invention provides a rapid detection method for microplastics in human intestinal tissue based on micro Raman spectroscopy. This method offers advantages such as efficient digestion of complex tissue samples, low-cost enrichment, simple and rapid detection, and identification of nanoscale microplastic particles, providing technical support for the qualitative and quantitative analysis of microplastics in human intestinal tissue samples.

[0005] The specific technical solution for achieving the objective of this invention is as follows:

[0006] A rapid detection method for microplastics in human intestinal tissue based on micro Raman spectroscopy, comprising the following specific steps:

[0007] Step 1: Alkaline solution digestion

[0008] Under water bath heating conditions, a 2 mol / L KOH solution was added to the human intestinal tissue sample for digestion; the water bath heating temperature was 50-60℃, and the amount of KOH solution added was 20-30 ml per 0.1-0.15 g of human intestinal tissue, with a maximum digestion time of 2 h;

[0009] Step 2: Neutralize and digest the solution

[0010] The digestion solution obtained in step 1 was neutralized to neutral using a 1 mol / L H2SO4 solution;

[0011] Step 3: Filter the digestion solution

[0012] The digestion solution was filtered onto the filter membrane using an all-glass frit filter, rinsed with ultrapure water, and then placed in a glass petri dish for testing; the filter membrane was a glass fiber filter membrane.

[0013] Step 4: Select representative areas

[0014] Select representative detection windows in the effective filtration area of ​​the filter membrane; use a 50x objective lens to select three detection windows and create a mosaic pattern. The three detection windows are: A located in the inner layer of the filter membrane; B located in the middle layer of the filter membrane; and C located at the edge of the filter membrane.

[0015] Step 5: Rapidly acquire Raman spectra

[0016] A panoramic clear image of the representative detection window in step 4 was obtained using a micro Raman spectrometer, and the image was then subjected to Raman spectroscopy.

[0017] Step 6: Determine the composition of the microplastics

[0018] The spectra collected in step 5 are compared with the standard spectral database to screen out polymers with a matching coefficient of at least 70, thus determining the composition of the microplastics; the database includes the built-in database of KnowItAll software and the Raman-Polymers-Wiley database;

[0019] Step 7; Count the total number of microplastics on the filter membrane.

[0020] The total number of microplastics on the filter membrane is obtained by calculating the concentration of microplastics in different representative detection windows, weighting them to the total area of ​​the corresponding filter membrane, and then summing the results.

[0021] Furthermore, the total number of microplastics on the filter membrane described in step 7 is calculated using the following formula:

[0022]

[0023] Wherein, N1, N2, and N3 are the number of microplastics identified within detection windows A, B, and C, respectively;

[0024] 'a' represents the area of ​​a single detection window, in mm. 2 ;

[0025] F1, F2, and F3 represent the abundance of microplastics per unit area within detection windows A, B, and C, respectively, in units of particles / mm². 2, ;

[0026] S W1 S W2 and S W3 These represent the areas of the inner layer W1, middle layer W2, and edge W3 of the effective filtration zone of the filter membrane, respectively, in mm. 2 ;

[0027] N total It represents the total number of microplastics on the filter membrane.

[0028] The beneficial effects of this invention are:

[0029] 1) This invention eliminates the need for staining markers, simplifying the analytical steps, and the sample can be used for subsequent analyses;

[0030] 2) The present invention establishes a method for rapid pretreatment of human intestinal tissue. Alkaline digestion in a water bath at 50-60℃ for a maximum of 2 hours can fully dissolve organic matter, which greatly simplifies the sample pretreatment method and can rapidly process batches of samples.

[0031] 3) The invention uses a glass fiber filter membrane, which has the advantages of low background interference, acid and alkali resistance and good mechanical strength. The cost of a single sheet is less than 0.5 yuan, which greatly reduces the detection cost. It can be used to replace the commonly used expensive imported silver filter membrane and is suitable for widespread promotion.

[0032] 4) This invention employs mosaic mosaic and panoramic clear imaging three-dimensional correction technology. If three representative detection windows (single window size: 1500×3500μm) are selected with a 50x objective lens, the detection window can account for 8.91% of the effective filter membrane area, which can meet the requirements of rapid positioning and efficient detection under a microplastic microscope.

[0033] In summary, this invention provides a rapid detection method for microplastics in human intestinal tissue using micro Raman spectroscopy. This method requires no staining or labeling, has simple analytical steps, low detection cost, and high efficiency. It can be used for batch testing of samples and can quickly achieve the extraction, qualitative, and quantitative detection of microplastics in human intestinal tissue. This method is of great significance for advancing research on the risk assessment of microplastic exposure in human intestinal tissue and its potential toxicological effects. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of microplastic extraction and Raman detection in human intestinal tissue samples in Example 1;

[0035] Figure 2 This is a schematic diagram showing the distribution of representative detection windows A, B, and C in the human intestinal wall tissue filter membrane sample in Example 1;

[0036] Figure 3 The images show the polyethylene (PE) Raman spectrum detected in the human intestinal wall tissue filter membrane sample in Example 1 and the PE standard spectrum from the KnowItAll software database.

[0037] Figure 4 The images show the Raman spectra of polyethylene terephthalate (PET) detected in the human intestinal wall tissue filter membrane sample in Example 1 and the standard PET spectra from the KnowItAll software database.

[0038] Figure 5 The images show the Raman spectra of polytetrafluoroethylene (PTFE) detected in the human intestinal wall tissue filter membrane sample in Example 1 and the standard PTFE spectra from the KnowItAll software database.

[0039] Figure 6 This is a schematic diagram showing the protein staining and decolorization of human intestinal wall tissue samples after different digestion times, acid neutralization, and filtration in Example 2. Detailed Implementation

[0040] The specific embodiments of the present invention will be described in further detail below.

[0041] The reagents and materials used are as follows: potassium hydroxide (analytical grade) and sulfuric acid (analytical grade) were purchased from Sinopharm Chemical Reagent Co., Ltd. Glass fiber filter membranes (φ=25 mm, pore size 0.22 μm) were purchased from Haining Delv New Material Technology Co., Ltd., and 0.25% Coomassie Brilliant Blue R-250 staining solution and 5% ethanol-10% glacial acetic acid destaining solution were purchased from Shanghai Nuoning Biotechnology Co., Ltd. The human intestinal tissue samples used in the examples were obtained from intestinal wall tissue surgically removed at a cancer hospital.

[0042] Example 1

[0043] See Figure 1 This embodiment provides a rapid detection method for microplastics in human intestinal tissue based on micro Raman spectroscopy, comprising the following steps:

[0044] (1) Establishment of human intestinal tissue sample pretreatment

[0045] like Figure 1As shown in Figure A, (a) accurately weigh 0.1 g of human intestinal wall tissue sample, repeatedly wash the surface impurities with ultrapure water, and drain for later use; (b) place it in a 50 ml Erlenmeyer flask and add 20 ml of 2 mol / L KOH solution; (c) place the Erlenmeyer flask in a 60℃ water bath for 2 h for digestion; (d) cool the digestion solution to room temperature and add 1 mol / L H2SO4 solution to neutralize the digestion solution to neutral; (e) filter the neutralized digestion solution using a 250 ml all-glass frit filter, allowing it to pass through a glass fiber filter membrane. Wash the Erlenmeyer flask three times with 60 ml of ultrapure water to ensure complete sample transfer, and filter the washing solution. Then rinse the glass fiber filter membrane with 10 ml of ultrapure water to further remove residual impurities; (f) remove the filter membrane and transfer it to a clean glass petri dish, air dry, and then seal and store at room temperature for testing.

[0046] (2) Select a representative area on the filter membrane

[0047] To balance analytical efficiency and representativeness, stratified randomized estimation was adopted. See the diagram below. Figure 2 The effective filtration radius of the filter membrane is r=7.5mm, and the total area is 176.71 mm². 2 It consists of three layers: inner, middle, and outer. The inner layer, W1, is a disk with a radius of r = 2.5 mm (area 19.64 mm²). 2 The middle layer W2 is a ring composed of r=2.5 mm and r=5.0 mm (area 58.90 mm²). 2 The outer layer W3 is a ring composed of r=5.0 mm and r=7.5 mm (area 98.17 mm²). 2 Three windows, A, B, and C, were set in the effective filtration area of ​​the glass fiber filter membrane, each with a size of 1500 × 3500 μm. Window A was located in the inner layer of the filter membrane (W1, 0°), representing the central region with a high concentration of microplastics; window B was located in the middle layer of the filter membrane (W2, 0°); and window C was located at the edge of the filter membrane (W3, 180°), representing the region with a relatively low concentration of microplastics. The total area of ​​the three windows was 15.75 mm², covering 8.91% of the effective filtration area.

[0048] (3) Micro Raman Detection of Microplastics

[0049] like Figure 1 As shown in Figure B, (g) detection was performed using a micro Raman spectrometer (Xplora Plus, HORIBA, FR); (h) Raman scattering tests were conducted on suspected microplastic particles in three representative areas using 50x objective mosaic and panoramic clear imaging, respectively. Test conditions: laser wavelength 785 nm, grating 1200, spectral scanning range 400-3000 cm⁻¹. -1Laser power 25%, spectral acquisition time 1 s, cumulative 5 times, slit 100μm, pinhole 300μm.

[0050] (4) Use KnowItAll software to determine the polymer composition.

[0051] like Figure 1 As shown in Figure B (i), the collected spectra were compared with the built-in databases (Raman-Biomaterials-Horiba and Raman-Forensic-Horiba databases) and the Raman-Polymers-Wiley database using KnowItAll software, and polymers with a matching coefficient exceeding 70 were screened. Analysis revealed that the microplastics present in the human intestinal wall tissue samples were polyethylene (PE), polyethylene terephthalate (PET), and polytetrafluoroethylene (PTFE). (See also...) Figure 3 Figure (a) shows the Raman spectrum of polyethylene (PE) detected in a human intestinal wall tissue filter sample, and (b) shows the standard PE spectrum from the KnowItAll software database; see reference. Figure 4 Figure (a) shows the Raman spectrum of polyethylene terephthalate (PET) detected in a human intestinal wall tissue filter sample, and (b) shows the standard PET spectrum from the KnowItAll software database; see reference. Figure 5 In the figure, (a) is the Raman spectrum of polytetrafluoroethylene (PTFE) detected in a human intestinal wall tissue filter membrane sample, and (b) is the standard spectrum of PTFE from the KnowItAll software database.

[0052] (5) Calculation method for the total number of microplastics on the filter membrane

[0053] The number of microplastics in three representative detection windows (A, B, and C) are counted, denoted as N1, N2, and N3 respectively. The abundance of microplastics per unit area in each layer is calculated as F1, F2, and F3 respectively. Finally, the number of microplastics in the three layers is weighted and summed according to the corresponding area to obtain the total number of microplastics on the entire filter membrane. The calculation formula is as follows:

[0054]

[0055] Wherein, N1, N2, and N3 are the number of microplastics identified in detection windows A, B, and C, respectively;

[0056] 'a' is the area of ​​a single detection window of 1500 × 3500 μm, in mm. 2 ;

[0057] F1, F2, and F3 represent the abundance of microplastics per unit area within detection windows A, B, and C, respectively, in units / mm². 2, ;

[0058] SW1 S W2 S W3 These are the areas of the inner layer W1, middle layer W2, and edge W3 of the effective filtration zone of the filter membrane, respectively, in mm. 2 ;

[0059] N total It represents the total number of microplastics in the effective filtration area of ​​the filter membrane.

[0060] Example 2

[0061] An optimization experiment on the digestion time of human intestinal wall tissue samples included the following steps:

[0062] (1) Digestion: Accurately weigh 0.1 g of human intestinal wall tissue sample, repeatedly wash its surface impurities with ultrapure water, and drain. Place the sample in a 50 ml Erlenmeyer flask, add 20 ml of 2 mol / L KOH solution, and then place the Erlenmeyer flask in a 60℃ water bath. Set four different digestion time groups (30 min, 60 min, 90 min, 120 min). After digestion, add 1 mol / L H2SO4 solution to the digestion solution until the digestion solution is neutral. Each digestion time group is repeated 3 times.

[0063] (2) Filtration: The digestion solution after neutralization in step (1) is filtered through a 250 ml all-glass frit filter to pass through a glass fiber filter membrane. The conical flask is washed three times with 60 ml of ultrapure water. The washing solution is filtered, and the filter membrane is rinsed with 10 ml of ultrapure water. The filter membrane is then removed and transferred to a clean glass petri dish.

[0064] (3) Staining: The four filter membranes with different digestion times obtained in step (2) were stained with 0.25% Coomassie Brilliant Blue R-250 staining solution on a horizontal shaker at 100 r / min for 5 min, and then the staining solution was poured out. After digestion, the protein only adhered to the surface of the filter membrane and did not penetrate into the internal pores. The protein could be fully bound by staining in a short time and a clear signal could be obtained. At the same time, the staining solution was difficult to wash off after binding with the filter membrane. In order to reduce the difficulty of destaining and avoid interference from residual solution, the staining time was set to 5 min in this experiment.

[0065] (4) Decolorization: Add 5% ethanol-10% glacial acetic acid decolorization solution to the stained filter membrane sample obtained in step (3) and wash it with shaking at 100 r / min for 15 min on a horizontal shaker. After washing, air dry the filter membrane and observe the staining differences of different digestion time groups.

[0066] As attached Figure 6As shown, in the four digestion time groups (30 min, 60 min, 90 min, and 120 min), after acid neutralization, filtration, staining, and destaining, the staining depth of the central filtration zone of the filter membrane exhibited a clear gradient change. With prolonged digestion time, the blue color of the central filtration zone gradually faded. Specifically, the 30 min group showed a distinct blue color in the central filtration zone, accompanied by a large amount of flocculent material; the blue color of the central filtration zone in the 60 min and 90 min groups weakened successively compared to the 30 min group, and the amount of flocculent material also gradually decreased; the 120 min group showed no obvious Coomassie Brilliant Blue staining signal in the central filtration zone, with only a very small amount of fine solid material observed. Due to the porous structure of the glass fiber membrane, there is some adsorption residue of Coomassie Brilliant Blue staining solution. Even after routine destaining, a small amount of staining solution may still adhere to the filter membrane surface, resulting in a stable dark blue background in the blank area of ​​the filter membrane.

[0067] The lightly stained central area in the 120-minute group was not caused by protein residue, but rather by the retention of alkali-resistant residual components from the intestinal wall tissue itself. These mainly include a small amount of fiber fragments remaining after digestion of intestinal wall collagen fibers, as well as insoluble precipitates formed by the reaction of minerals within intestinal wall cells with digestive juices. These residual components occupy staining sites on the filter membrane, making it difficult for Coomassie Brilliant Blue staining solution to be fully adsorbed in this area, even though it tends to remain on the filter membrane, resulting in light staining. This phenomenon is a normal experimental result and does not affect the retention efficiency of microplastics or subsequent detection. On the contrary, it indicates that interfering components such as proteins have been completely removed, and microplastics can be precisely retained.

[0068] Therefore, digesting a 0.1 g human intestinal wall tissue sample with 20 ml of 2 mol / L KOH solution at 60°C for 2 hours can completely remove interfering components and effectively retain microplastics.

Claims

1. A rapid detection method for microplastics in human intestinal tissue based on micro Raman spectroscopy, characterized in that, The method includes the following specific steps: Step 1: Alkaline solution digestion Under water bath heating conditions, a 2 mol / L KOH solution was added to the human intestinal tissue sample for digestion; the water bath heating temperature was 50-60℃, and the amount of KOH solution added was 20-30 ml per 0.1-0.15 g of human intestinal tissue, with a maximum digestion time of 2 h; Step 2: Neutralize and digest the solution The digestion solution obtained in step 1 was neutralized to neutral using a 1 mol / L H2SO4 solution; Step 3: Filter the digestion solution The digestion solution was filtered onto the filter membrane using an all-glass frit filter, rinsed with ultrapure water, and then placed in a glass petri dish for testing; the filter membrane was a glass fiber filter membrane. Step 4: Select representative areas Select representative detection windows in the effective filtration area of ​​the filter membrane; use a 50x objective lens to select three detection windows and create a mosaic pattern. The three detection windows are: A located in the inner layer of the filter membrane; B located in the middle layer of the filter membrane; and C located at the edge of the filter membrane. Step 5: Rapidly acquire Raman spectra A panoramic clear image of the representative detection window in step 4 was obtained using a micro Raman spectrometer, and the image was then subjected to Raman spectroscopy. Step 6: Determine the composition of the microplastics The spectra collected in step 5 are compared with the standard spectral database to screen out polymers with a matching coefficient of at least 70, thus determining the composition of the microplastics; the database includes the built-in database of KnowItAll software and the Raman-Polymers-Wiley database; Step 7; Count the total number of microplastics on the filter membrane. The total number of microplastics on the filter membrane is obtained by calculating the concentration of microplastics in representative detection windows, weighting them to the total area of ​​the corresponding filter membrane, and then summing the results.

2. The rapid detection method according to claim 1, characterized in that, The total number of microplastics on the filter membrane mentioned in step 7 is calculated using the following formula: ； Wherein, N1, N2, and N3 are the number of microplastics identified within detection windows A, B, and C, respectively; 'a' represents the area of ​​a single detection window, in mm. 2 ; F1, F2, and F3 represent the abundance of microplastics per unit area within detection windows A, B, and C, respectively, in units of particles / mm². 2, ; S W1 S W2 and S W3 These represent the areas of the inner layer W1, middle layer W2, and edge W3 of the effective filtration zone of the filter membrane, respectively, in mm. 2 ; N total It represents the total number of microplastics on the filter membrane.

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