A convenient detection method for microplastics based on composite fluorescent staining

Abstract

The present invention provides a kind of microplastic convenient detection method based on composite fluorescent staining, belong to the field of environmental pollutant detection. The method comprises: step one, using hydrogen peroxide with a concentration of 30% to digest the water sample to be detected to obtain a first solution; step two, the first solution is filtered with a filter membrane, microplastics are trapped on the filter membrane, and then rinsed with clean water to obtain the filter membrane after rinsing; step three, 10mL-50mL of a mixed dye solution of Nile red and 4',6-diamidino-2-phenylindole is added to the rinsed filter membrane, after standing for 30min under dark conditions, the dye solution is filtered to obtain a filter membrane with microplastics trapped after dyeing; step four, the filter membrane obtained in step three is transferred to a clean glass culture dish, placed at 45°C and dried for 12h to obtain a dried filter membrane; step five, under a predetermined exposure time, the dried filter membrane is observed and counted using a fluorescence microscope to obtain the content of microplastics in the water sample to be detected.

Description

Technical Field

[0001] The present invention belongs to the field of environmental pollutant detection, and specifically relates to a convenient microplastic detection method based on composite fluorescent staining. Background Art

[0002] Microplastics are ubiquitous in natural environments, including water, soil, and the atmosphere. They have even been detected in polar snow, food, and animal tissue. Microplastic pollution has become a major environmental concern in my country and globally. Microplastics can not only damage organisms through their physical effects, but more importantly, their environmental persistence, mobility, and ability to accumulate pollutants make them a significant vector for the spread of other pollutants, posing a serious threat to biodiversity, ecological security, and human health. Both the Chinese government and the scientific community are paying increasing attention to the microplastics issue. The March 5, 2022, National Government Work Report clearly stated the need to strengthen the control of emerging pollutants. Subsequently, the Ministry of Ecology and Environment further clarified microplastics as one of four emerging pollutants and issued an action plan for the control of emerging pollutants, incorporating new pollutants such as microplastics into the environmental management system. This means that microplastic detection will reach a peak and remain so for an extended period. Therefore, developing an efficient, reliable, convenient, and low-cost microplastic detection technology is crucial for ensuring stricter microplastic management in the future.

[0003] In recent years, domestic and foreign scholars have developed and reported a variety of microplastic analysis and detection technologies, which can be summarized into four types: visual method, fluorescent staining method, spectroscopy and mass spectrometry. Each method has its own characteristics. The visual method is simple to operate, but it is only suitable for the quantitative detection of microplastics with a particle size greater than 100 microns; the fluorescent staining method is also relatively convenient, and the applicable microplastic particle size can reach several microns, but the detection process is easily interfered by organic matter; spectroscopy (micro-infrared spectroscopy or micro-Raman spectroscopy) and mass spectrometry can both perform quantitative and qualitative analysis of microplastics with high accuracy, but they rely on expensive instruments and equipment, and require spectral comparison or analysis, and the detection takes a long time. Faced with the increasing demand for microplastic detection, fluorescent staining has obvious advantages. How to overcome the problem of organic matter interference is the key to promoting the widespread application of fluorescent staining. Summary of the Invention

[0004] The present invention is conducted to solve the above-mentioned problems, and its purpose is to provide a convenient microplastic detection method based on composite fluorescent staining.

[0005] The present invention provides a convenient detection method for microplastics based on composite fluorescent staining, which has the following characteristics: step 1, using 30% hydrogen peroxide as a digestion solution, mixing the digestion solution with a water sample to be detected, and then digesting it at high temperature to obtain a first solution; step 2, filtering the first solution through a clean filter membrane to extract the microplastics in the first solution, and then rinsing the filter membrane with clean water and vacuum drying, and repeating the rinsing operation twice to obtain a rinsed filter membrane; step 3, adding 10mL of -50mL of a mixed dye solution of Nile red and 4',6-diamidino-2-phenylindole is placed in darkness for staining for 30 minutes, and then excess dye solution is removed by vacuum filtration to obtain a filter membrane that retains the stained microplastics; Step four, the filter membrane that retains the stained microplastics is transferred to a clean glass culture dish, covered with a lid, and dried at 45°C for 12 hours to obtain a dry filter membrane; Step five, under a predetermined exposure time, the dry filter membrane is observed using a fluorescence microscope, and the partitioning statistics method is used to count the microplastics to obtain the content of microplastics in the water sample to be tested.

[0006] The convenient microplastic detection method based on composite fluorescent staining provided by the present invention may also have the following characteristics: wherein, in step one, the volume ratio of the digestion solution to the water sample to be detected is 1:1 to 50.

[0007] The convenient microplastic detection method based on composite fluorescent staining provided by the present invention may also have the following characteristics: wherein, in step one, digestion at high temperature refers to digestion at 50°C-100°C for 5h-24h.

[0008] The convenient microplastic detection method based on composite fluorescent staining provided by the present invention may also have the following characteristics: wherein, in step 2, the filter membrane is a glass fiber filter membrane, which is calcined at 500°C for 1h-2h before use to remove background microplastic interference.

[0009] The convenient microplastic detection method based on composite fluorescent staining provided by the present invention may also have the following characteristics: wherein, in step 2, 20 mL of clean water is added for each rinse, and the clean water is any one of distilled water filtered through a glass fiber filter membrane, bottled water, RO pure water, and ultrapure water.

[0010] The convenient microplastic detection method based on composite fluorescent staining provided by the present invention may also have the following characteristics: wherein, in step three, the working concentrations of Nile red and 4',6-diamidino-2-phenylindole in the mixed dye during staining are 5 mg / L-20 mg / L and 0.2 mg / L-2 mg / L, respectively.

[0011] The convenient microplastic detection method based on composite fluorescent staining provided by the present invention may also have the following characteristics: wherein, in step four, the clean glass culture dish has been subjected to a calcination treatment at 500°C for 1 hour.

[0012] The convenient microplastic detection method based on composite fluorescent staining provided by the present invention may also have the following characteristics: wherein, in step five, the predetermined exposure time is 5ms-20ms, and when observing using a fluorescence microscope, first observe the particles stained with Nile red under the parameter setting of an excitation wavelength of 510-560nm and an emission wavelength >520nm; then observe the particles stained with DAPI under the parameter setting of an excitation wavelength of 330-380nm and an emission wavelength >420nm, where only the particles stained with Nile red are microplastics.

[0013] The convenient microplastic detection method based on composite fluorescent staining provided by the present invention may also have the following characteristics: when observing with a fluorescence microscope, if the magnification is 40×, the predetermined exposure time is 10ms-20ms; if the magnification is 100×, the predetermined exposure time is 5ms-10ms.

[0014] The convenient microplastic detection method based on composite fluorescent staining provided by the present invention may also have the following characteristics: wherein, the partition statistics method refers to randomly selecting 5 partitions of known area on the dry filter membrane, counting them separately, and then calculating the total number of microplastics N using the following formula:

[0015]

[0016] The following formula is used to calculate the content C of microplastics in the water sample to be tested:

[0017]

[0018] Where V represents the volume of the water sample to be tested.

[0019] Compared with the prior art, the beneficial effects of the present invention are embodied in:

[0020] 1. Solved the problem that the single Nile red staining method is limited by organic matter interference

[0021] Although the single Nile red staining method is convenient, since Nile red dye can not only stain microplastics but also many other organic particles, this method is easily interfered with by organic matter and is greatly limited in its application. Although studies have used different methods to pretreat samples to remove organic matter, organic matter of microbial origin is difficult to effectively remove. The method of the present invention effectively eliminates the influence of organic matter in the sample through hydrogen peroxide digestion and co-staining with Nile red and 4',6-diamidino-2-phenylindole (DAPI).

[0022] 2. The detection accuracy of microplastic size is guaranteed by optimizing the fluorescence observation exposure time

[0023] The harmfulness of microplastics is not only related to their morphology, but also closely linked to their size. When using fluorescent staining to detect microplastics, excessively long exposure times can significantly overestimate their size, while insufficient exposure times can prevent the detection of some small microplastics. This invention, through continuous optimization and testing of exposure time, ensures excellent detection accuracy of observed microplastic size, with a discrepancy of <10% from actual size.

[0024] 3. Combines convenience and accuracy

[0025] Spectroscopy (including micro-Raman and micro-infrared) and mass spectrometry are currently generally considered to be microplastic detection methods with high detection accuracy, but they must rely on expensive instruments and relatively complex analysis processes. The analysis and detection process of fluorescent staining is more convenient, but a single fluorescent staining method usually has insufficient detection accuracy. The composite fluorescent staining method disclosed in the present invention not only maintains the inherent convenience of fluorescent staining, but also significantly improves its detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a graph showing changes in observed sizes of microplastics at different exposure times in Example 2 of the present invention. Figure 1 a1-a5 represent the results of exposure times of 5ms, 10ms, 20ms, 50ms, and 100ms at 40× magnification, respectively. Figure 1 b1-b5 represent the results at 100× magnification with exposure times of 5ms, 10ms, 20ms, 50ms, and 100ms, respectively;

[0027] Figure 2 This is a graph showing the recovery rates of microplastics of different sizes detected by the convenient microplastic detection method based on composite fluorescent staining in Example 3 of the present invention;

[0028] Figure 3 This is a visual field diagram of the convenient microplastic detection method based on composite fluorescent staining under two fluorescence observation conditions in Example 3 of the present invention;

[0029] Figure 4 This is a comparison chart of the convenient microplastic detection method based on composite fluorescent staining in Example 3 of the present invention and the commonly used Nile red staining method and micro-Raman method. DETAILED DESCRIPTION

[0030] The specific principles of the method of the present invention are as follows:

[0031] Nile red dye stains not only microplastics but also many other organic particles. Therefore, single Nile red staining is only suitable for detecting microplastics in water samples with very low organic particle content. Water sample digestion methods can effectively remove large amounts of organic matter, but are less effective at removing organic solids of microbial origin, which often contain DNA. DAPI cannot stain microplastics, but it can bind to DNA and stain it, thus eliminating interference from organic solids of microbial origin. Ultimately, solid matter that is stained by Nile red but not by DAPI is microplastics.

[0032] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the following examples and drawings specifically illustrate the convenient microplastic detection method based on composite fluorescent staining of the present invention.

[0033] <Example 1>

[0034] Example 1 provides a convenient method for detecting microplastics based on composite fluorescent staining, which includes the following steps:

[0035] Step 1: Use 30% hydrogen peroxide as a digestion solution, mix the digestion solution with the water sample to be tested in a volume ratio of 1:10, and then digest at 50° C. for 24 hours to obtain a first solution.

[0036] Step 2: Filter the first solution through a clean filter membrane. The microplastics in the first solution are retained on the filter membrane. The filter membrane is then rinsed with 20 mL of clean water and vacuum-dried. This rinse process is repeated twice to obtain a rinsed filter membrane. The filter membrane is a glass fiber filter membrane, which is calcined at 500°C for 1-2 hours before use to remove background microplastic interference. Clean water can be any of distilled water, bottled water, RO purified water, and ultrapure water filtered through a glass fiber filter membrane.

[0037] Step 3: Add 10 mL of a mixed solution of Nile red and 4',6-diamidino-2-phenylindole to the rinsed filter membrane, place it in the dark, let it stand for 30 minutes, and then vacuum filter to remove excess dye to obtain a filter membrane that retains the stained microplastics. The working concentrations of Nile red and DAPI in the mixed dye are 10 mg / L and 0.5 mg / L, respectively.

[0038] Step 4: Transfer the filter membrane containing the dyed microplastics to a clean glass petri dish, cover it, and dry it at 45°C for 12 hours to obtain a dry filter membrane. The clean glass petri dish has been calcined at 500°C for 1 hour.

[0039] Step 5: Observe the dried filter membrane using a fluorescence microscope under a predetermined exposure time, and count using the partitioning statistics method to obtain the content of microplastics in the water sample to be tested.

[0040] In step 5, the predetermined exposure time is 5ms-20ms. When observing with a fluorescence microscope, if the magnification is 40×, the predetermined exposure time is 10ms-20ms; if the magnification is 100×, the predetermined exposure time is 5ms-10ms.

[0041] When observing using a fluorescence microscope, first observe the particles stained with Nile red under the parameter settings of excitation wavelength of 510-560nm and emission wavelength >520nm (red fluorescence); then observe the particles stained with DAPI under the parameter settings of excitation wavelength of 330-380nm and emission wavelength >420nm (blue fluorescence), among which only the particles stained with Nile red are microplastics.

[0042] The partition statistics method refers to randomly selecting 5 partitions of known area on a dry filter membrane, counting them separately, and then calculating the total number of microplastics N using the following formula:

[0043]

[0044] In the formula, the total amount of microplastics in each partition is the difference between the counts of the partition under the above-mentioned red fluorescence and blue fluorescence.

[0045] The following formula is used to calculate the content C of microplastics in the water sample to be tested:

[0046]

[0047] Where V represents the volume of the water sample to be tested.

[0048] <Example 2>

[0049] Example 2 is to illustrate the effect of exposure time optimization on the accuracy of microplastic size detection in the convenient microplastic detection method based on composite fluorescent staining of the present invention.

[0050] In Example 2, microplastic particles of two sizes, 5 μm and 20 μm, dyed with Nile red, were used as model microplastics. A random amount of the model microplastics was placed in a clean glass cuvette, covered, and dried at 45°C for 12 hours. The sample was then observed under a fluorescence microscope. Five gradient exposure times were set for each microplastic size: 5 ms, 10 ms, 20 ms, 50 ms, and 100 ms. Software was used to calculate the observed microplastic size. Observation results at 40× and 100× magnifications were also compared.

[0051] Figure 1 This is a graph showing changes in observed sizes of microplastics at different exposure times in Example 2 of the present invention. Figure 1 a1-a5 represent the results of exposure times of 5ms, 10ms, 20ms, 50ms, and 100ms at 40× magnification, respectively. Figure 1 b1-b5 in the figure represent the results at a magnification of 100× with exposure times of 5ms, 10ms, 20ms, 50ms, and 100ms, respectively.

[0052] Depend on Figure 1 It can be seen that the observed size of microplastics of both particle sizes increases with the increase of exposure time. When the magnification is 40× ( Figure 1 In a1-a5), when the exposure time is less than 20ms, there are differences of 0.0-8.7% and 1.1%-5.7% between the observed sizes of 5μm and 20μm microplastics and the actual sizes, respectively. However, when the exposure time is 5ms, the number of microplastics in the field of view is significantly less than that of other exposure times. Once the exposure time reaches 50ms and above, the edges of the observed microplastics (especially 20μm microplastics) become blurred, the size increases significantly, and it is difficult to count accurately. Therefore, when the magnification is 40×, 10-20ms is the recommended exposure time.

[0053] When the magnification is 100× ( Figure 1 In items b1-b5), when the exposure time is less than 10ms, the observed size of 5μm and 20μm microplastics differ from their actual sizes by 1.0%-6.0% and 2.2%-4.7%, respectively. When the exposure time reaches 20ms, the observed size of both microplastic particle sizes exceeds the actual size by more than 12%. Further increases in exposure time lead to blurred vision and uncountable microplastics. Therefore, at a magnification of 100×, an exposure time of 5-10ms is recommended.

[0054] <Example 3>

[0055] Example 3 is intended to illustrate the principle and detection reliability of the convenient microplastic detection method based on composite fluorescent staining of the present invention.

[0056] Example 3: First, the two Nile red-stained microplastics of Example 2 were used as model microplastics to test the recovery rate of the convenient microplastic detection method based on composite fluorescent staining of the present invention through a spike recovery experiment. For each microplastic particle size, three microplastic spike levels were set (<100, 200-300, and 400-500).

[0057] The convenient microplastic detection method based on composite fluorescent staining described in Example 1 was then used to test the microplastic content in laboratory tap water, surface water from a site in Hefei, and effluent from a sewage treatment plant in Hefei. The results were compared with commonly used Nile red staining and micro-Raman methods. The glassware and filter membranes used in water sampling and laboratory analysis were calcined at 500°C for 1 hour, and the purified water used in the experiments was filtered.

[0058] When the convenient microplastic detection method based on composite fluorescent staining was used to test the content of microplastics in laboratory tap water, surface water from a certain area in Hefei, and effluent from a sewage treatment plant in Hefei, the three water sources were used as a water sample to be tested. Each water sample to be tested was tested according to the following process:

[0059] Step 1: add 20 mL of 30% hydrogen peroxide to 200 mL of the water sample to be tested, and then digest it at 50° C. for 24 hours to obtain a first solution.

[0060] Step 2: Filter the first solution through a glass fiber filter membrane with a pore size of 0.7 μm. At this time, the microplastics are retained on the filter membrane. Then rinse the filter membrane with 20 mL of clean water and vacuum dry it. Repeat the rinsing operation twice (a total of 3 times) to obtain the rinsed filter membrane.

[0061] Step 3: Add 10 mL of mixed dye solution to the rinsed filter membrane, place it in the dark, let it stand for 30 minutes, and then vacuum filter to remove excess dye solution to obtain a filter membrane that has retained the stained microplastics. The mixed dye solution is a mixed dye solution of Nile red and DAPI, and the concentrations of Nile red and DAPI in the mixed dye solution are 10 mg / L and 0.5 mg / L, respectively.

[0062] Step 4: Transfer the filter membrane that has retained the dyed microplastics to a clean glass culture dish, cover it with a lid, and dry it at 45°C for 12 hours to obtain a dry filter membrane.

[0063] Step 5: Observe the dried filter membrane using a fluorescence microscope at an exposure time of 10ms and a magnification of 100×, and count using the partitioning statistical method to obtain the content of microplastics in the water sample to be tested.

[0064] Among them, when using a fluorescence microscope for observation, the particles stained with Nile red are first observed under the parameter settings of excitation wavelength of 510-560nm and emission wavelength >520nm (red fluorescence); then the particles stained with DAPI are observed under the parameter settings of excitation wavelength of 330-380nm and emission wavelength >420nm (blue fluorescence), among which only the particles stained with Nile red are microplastics.

[0065] The partition statistics method refers to randomly selecting 5 partitions of known area on a dry filter membrane, counting them separately, and then calculating the total number of microplastics N using the following formula:

[0066]

[0067] In the formula, the total amount of microplastics in each partition is the difference between the counts of the partition under the above-mentioned red fluorescence and blue fluorescence.

[0068] The following formula is used to calculate the content C of microplastics in the water sample to be tested:

[0069]

[0070] Where V represents the volume of the water sample to be tested.

[0071] Figure 2 This is a recovery rate graph of microplastics of different sizes detected by the convenient microplastic detection method based on composite fluorescent staining in Example 3 of the present invention.

[0072] Depend on Figure 2 It can be seen that at all tested spike levels, the recovery rates of microplastics of both particle sizes remained between 90% and 97%, proving the reliability of the convenient microplastic detection method based on composite fluorescent staining of the present invention.

[0073] Figure 3 This is a visual field diagram of the convenient microplastic detection method based on composite fluorescent staining under two fluorescence observation conditions in Example 3 of the present invention.

[0074] Figure 3The microscope field of view of the composite fluorescent-stained sample under two different fluorescence observation conditions is shown. It can be clearly seen that under the condition of Ex / Em = 510-560nm / >590nm, many red fluorescent particles appear, namely particles stained with Nile red. When the observation condition is switched to Ex / Em = 330-380nm / >420nm, a considerable portion of the particles at the corresponding positions of the Nile red-stained particles appear light blue, that is, they are stained with DAPI, indicating that the particles are not microplastics, but organic solids of microbial origin. This shows that composite fluorescent staining effectively overcomes the interference of microbial organic matter on microplastic detection.

[0075] Figure 4 This is a comparison chart of the convenient microplastic detection method based on composite fluorescent staining in Example 3 of the present invention and the commonly used Nile red staining method and micro-Raman method.

[0076] like Figure 4 As shown, the microplastic content in tap water, surface water, and effluent from a sewage treatment plant measured by the single Nile red staining method was 3535 pieces / L, 15308 pieces / L, and 16123 pieces / L, respectively; the microplastic content in the three water samples detected by the method disclosed in the present invention was 1065 pieces / L, 2948 pieces / L, and 3939 pieces / L, respectively, which was more than 70% lower than the result of the single Nile red staining method. Moreover, compared with the micro-Raman method, which has very high accuracy, the difference in microplastic content of all samples did not exceed 15%, proving that the convenient microplastic detection method based on composite fluorescent staining of the present invention has good accuracy.

[0077] The above embodiments are preferred examples of the present invention and are not intended to limit the scope of protection of the present invention.

Claims

1. A convenient microplastic detection method based on composite fluorescent staining, characterized in that: The following steps are involved: Step 1: using 30% hydrogen peroxide as a digestion solution, mixing the digestion solution with the water sample to be tested, and then digesting it at a high temperature to obtain a first solution; Step 2: The first solution is filtered through a clean filter membrane to extract the microplastics in the first solution. The microplastics in the first solution are retained on the filter membrane. The filter membrane is then rinsed with clean water and vacuum-dried. The rinsing operation is repeated twice to obtain a rinsed filter membrane. Step 3: Add 10 mL to 50 mL of a mixed dye solution of Nile red and 4',6-diamidino-2-phenylindole to the rinsed filter membrane, place it in the dark and dye it for 30 minutes, and then vacuum filter to remove excess dye solution to obtain a filter membrane that retains the dyed microplastics; Step 4: Transfer the filter membrane containing the dyed microplastics to a clean glass culture dish, cover it with a lid, and dry it at 45°C for 12 hours to obtain a dry filter membrane; Step 5: Observe the dried filter membrane using a fluorescence microscope under a predetermined exposure time, and count using a partitioning statistical method to obtain the content of microplastics in the water sample to be tested. Wherein, in step 1, the volume ratio of the digestion solution to the water sample to be tested is 1: (1-50), and digestion at high temperature means digestion at 50°C-100°C for 5h-24h, In step 3, the working concentrations of Nile red and 4',6-diamidino-2-phenylindole in the mixed dye during dyeing are 5 mg / L-20 mg / L and 0.2 mg / L-2 mg / L, respectively. In step 5, the predetermined exposure time is 5ms-20ms. When observing using the fluorescence microscope, first observe the particles stained with Nile red under the parameter setting of excitation wavelength of 510-560 nm and emission wavelength>520 nm; then observe the particles stained with DAPI under the parameter setting of excitation wavelength of 330-380 nm and emission wavelength>420 nm. The particles stained only with Nile red are microplastics. When observing with the fluorescence microscope, if the magnification is 40×, the predetermined exposure time is 10ms-20ms; if the magnification is 100×, the predetermined exposure time is 5ms-10ms.

2. The convenient microplastic detection method based on composite fluorescent staining according to claim 1 is characterized in that: in, In step 2, the filter membrane is a glass fiber filter membrane, which is calcined at 500°C for 1h-2h before use to remove background microplastic interference.

3. The convenient microplastic detection method based on composite fluorescent staining according to claim 1 is characterized in that: in, In step 2, add 20 mL of the clean water each time you rinse. The clean water is any one of distilled water, bottled water, RO pure water and ultrapure water filtered through a glass fiber filter membrane.

4. The convenient microplastic detection method based on composite fluorescent staining according to claim 1 is characterized in that: in, In step 4, the clean glass culture dish has been calcined at 500° C. for 1 hour.

5. The convenient microplastic detection method based on composite fluorescent staining according to claim 1 is characterized in that: in, The partitioning statistical method refers to randomly selecting 3-7 partitions of known area on the dry filter membrane, counting them separately, and then calculating the total number of microplastics N using the following formula: The following formula is used to calculate the content C of microplastics in the water sample to be tested: Where V represents the volume of the water sample to be tested.

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