Nano-plastic content detection method based on Py-GCMS technology
Through Py-GC/MS technology combined with chemical reagent extraction, the problem of nanoplastic detection in microorganisms is solved, quantitative analysis is realized, and scientific basis for the impact on nanoplastic ecology and health.
Patent Information
- Application Number
- CN202411727255.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-28
- Publication Date
- 2025-02-28
AI Technical Summary
The existing technology is difficult to effectively detect and quantitatively analyze the content of nanoplastics in microorganisms, resulting in a lack of data support for research on their ecological and health effects.
Using nanoplastic content detection method based on Py-GC/MS technology, the cell wall and protoplasts of E. coli were isolated and extracted, and protein crowns were formed by combining chemical reagents such as BSA and HNO3, and quantitative analysis was performed using Py-GC/MS.
Accurate measurement of the content of nanoplastics in microorganisms has been achieved, revealing the impact of nanoplastics on microorganisms, and providing new tools and methods for environmental monitoring and ecological effect assessment.
Smart Images
Figure 00000000_0000_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of nanoplastic detection, and specifically relates to a nanoplastic content detection method based on Py-GCMS technology. Background Art
[0002] As global plastic production continues to rise, plastic pollution has become an increasingly serious environmental problem. According to statistics, millions of tons of plastic waste are discharged into the environment every year, most of which eventually flow into the ocean, causing profound impacts on marine life and ecosystems. The terrestrial environment is no exception. The widespread distribution of plastic waste poses a potential threat to soil quality, terrestrial biodiversity and human health. Of particular concern is that the behavior and impact of nanoplastics produced during the decomposition of plastics in the environment are not fully understood. Nanoplastics are easily absorbed by organisms due to their extremely small size, and may penetrate the cell walls and cell membranes of organisms, enter the interior of the cells, and interfere with the normal function of the cells.
[0003] However, despite the widespread concern about the potential risks of this new type of pollutant, current research on nanoplastics mainly focuses on their environmental distribution, bioaccumulation, and impacts on higher organisms, while relatively little research has been done on the behavior and impacts of nanoplastics in microorganisms. As a fundamental component of an ecosystem, the health status and biological activity of microorganisms play a vital role in the stability and function of the entire ecosystem. Therefore, a deep understanding of the impact of nanoplastics on microorganisms is crucial to assessing the health of the entire ecosystem.
[0004] In view of the gaps in current research, the present invention aims to propose a method based on Py-GC / MS technology for analyzing and quantifying the content of nanoplastics in microorganisms, aiming to make up for the limitations of existing technologies in this field, help to deeply understand the potential ecological and health effects of nanoplastics, and provide environmental scientists with a new tool and method to more accurately monitor and evaluate the ecological effects of nanoplastic pollution, and provide a scientific basis for the formulation of environmental protection measures and related policies.
[0005] In this regard, the inventors proposed a nanoplastic content detection method based on Py-GCMS technology to solve the above problems. Summary of the invention
[0006] The object of the present invention is to provide a method for detecting nanoplastic content based on Py-GCMS technology to solve the problems raised in the above background technology.
[0007] To achieve the above object, the present invention provides the following technical solutions:
[0008] A method for detecting nanoplastic content based on Py-GCMS technology, comprising:
[0009] S1. Expand the culture of E. coli from a single clone;
[0010] S2, separating the cell wall of the Escherichia coli obtained in step S1, and centrifuging to obtain a supernatant and a precipitate;
[0011] S3, extracting nanoplastics from the supernatant obtained in step S2;
[0012] S4, extracting nanoplastics from the precipitate obtained in step S2;
[0013] S5. Use Py-GC / MS to quantitatively analyze the nanoplastics in steps S3 and S4;
[0014] S6. Analyze the effect of nanoplastics on Escherichia coli;
[0015] S7. Conduct a quantitative experiment on the adsorption and uptake of nanoplastics by Escherichia coli and optimize it according to different experimental conditions.
[0016] Preferably, the method for expanding the culture of Escherichia coli from a monoclonal colony in step S1 is: picking a monoclonal Escherichia coli colony on LB solid agar medium and inoculating it into 5 mL of LB liquid medium, shaking and culturing the inoculated medium at 37°C and 200 rpm overnight, and diluting the bacterial solution with the medium to an OD of 600 The diluted bacterial solution was transferred to a centrifuge tube and centrifuged at 4°C, 3000g for 20 min. The supernatant was discarded, the bacterial cell pellet was collected, and the bacteria were washed twice with 10mM Tris-HCl buffer, pH 8.0, to remove the residual culture medium.
[0017] Preferably, in step S2, the cell wall of Escherichia coli is separated, the washed bacteria are suspended in Tris-HCl buffer with a pH of 8.0, EDTA is added to a final concentration of 10 mM, and the mixture is shaken to allow EDTA to complex with the bacterial cell wall. The mixture is centrifuged again at 4000g, 4°C, 20min to remove the supernatant, 2 mg / mL of lysozyme is added, and the mixture is shaken at 37°C and 100 rpm for 30 minutes to further degrade the cell wall. Finally, the mixture is centrifuged again to obtain a supernatant and a precipitate.
[0018] Preferably, the supernatant is a cell wall component, and the precipitate is Escherichia coli without a cell wall, i.e., protoplasts.
[0019] Preferably, the method for extracting nanoplastics from the supernatant in step S3 is: take 40 mL of the supernatant containing cell walls in step S2, add 1 μg of synthesized nanoplastics, and add 0.2 mL of 4 g / L BSA solution thereto, use an orbital shaker, oscillate at 300 rpm for 5 minutes to promote the binding of nanoplastics and BSA to obtain a binding solution; use diluted HNO3 to adjust the pH of the binding solution to 4.0, heat it in a 60°C water bath for 10 minutes, so that the protein and the nanoplastic form a protein corona, after cooling, centrifuge at 8000 rpm for 10 minutes, precipitate the protein corona containing nanoplastics, and redisperse the protein corona in 200 μL of ultrapure water, transfer it to an 80 μL thermal lysis sample cup, and dry it at 100°C for 10 minutes.
[0020] Preferably, the method for extracting nanoplastics from the precipitate in step S4 is: adding 1 μg of synthetic nanoplastics to the precipitate containing the protoplast sample in step S2, and then adding 200 μL of H2O2 solutions of different concentrations to achieve the digestion of the protoplasts and release the nanoplastics therein, and transferring the treated solution to a thermal pyrolysis sample cup for thermal pyrolysis and quantitative analysis of the nanoplastics.
[0021] Preferably, the quantitative analysis method in step S5 is: using Py-GC / MS for quantitative analysis of nanoplastics, wherein the working parameters include pH value, BSA concentration and temperature, which are optimized according to the experimental requirements, and each sample is repeated three to five times, wherein the pH range is 2.5-4.5, the BSA concentration is 0-80 mg / L, the temperature is 40-80°C, and the spiked recovery rate is determined.
[0022] Preferably, the expression of the spiked recovery is:
[0023]
[0024] Among them, C e , unit μg / L, is the concentration of nanoplastics measured in a sample with a known added nanoplastic concentration of C, unit μg / L, while C0, unit μg / L, is the concentration of nanoplastics detected in a sample without added nanoplastics.
[0025] Preferably, the method for analyzing the effect of nanoplastics on Escherichia coli in step S6 is: adding PS-SO3H-NPs and PS-NH2-NPs to LB medium, setting up a blank control group, culturing and measuring OD according to the above method 600 , determining the effects of different nanoplastics on the growth of Escherichia coli.
[0026] Preferably, the method of the quantitative experiment in step S7 is to inoculate Escherichia coli into 40 mL of LB medium at a ratio of 1:100, culture for 12 hours, and then centrifuge at 4000 rpm for 10 minutes to collect the cells. Subsequently, the bacterial cells are washed twice with PBS to remove the residues in the culture medium. After washing, the cells are resuspended in 50 mL of PBS, and 5 μg of synthetic nanoplastic is added to the cell suspension, and shaken at 37°C and 200 rpm for 6 hours to promote the interaction between the nanoplastic and Escherichia coli.
[0027] Compared with the prior art, the present invention has the following beneficial effects:
[0028] (1) The present invention provides a novel method based on Py-GC / MS technology for quantitative determination of nanoplastics in Escherichia coli. Chemical reagents such as Tris-HCl, EDTA and lysozyme are used to effectively separate the cell wall and protoplasts of Escherichia coli, and Py-GC / MS analysis is performed under optimized experimental conditions. The content of nanoplastics in these samples can be accurately determined. In addition, the effectiveness of this method is verified by exposing newly cultured Escherichia coli to phosphate buffered solution (PBS) containing nanoplastics.
[0029] (2) The nanoplastics synthesized in the present invention have little effect on the growth of E. coli, and also reveal the different absorption characteristics of E. coli cell wall and protoplasts to nanoplastics. In particular, positively charged PS-NH2-NPs are easier to attach to the E. coli cell wall than negatively charged PS-SO3H-NPs, which indicates that it is related to the charge characteristics of E. coli itself. In addition, the key factors affecting the extraction efficiency of nanoplastics were systematically optimized, including BSA concentration, sample pH value and water bath temperature, to ensure the effective formation of protein corona and stable extraction of nanoplastics.
[0030] (3) The present invention provides a new tool and method to monitor and evaluate the ecological effects of nanoplastics in environmental samples, and provides new insights into the potential impact of nanoplastics on microbial communities. The method exhibits high accuracy, stability, and repeatability, providing a scientific basis for future environmental monitoring. It is of great significance for a deep understanding of the behavior of nanoplastics in the environment and their impact on microorganisms. It also provides a new analytical method for the field of environmental science. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Figure 1 This is a flow chart of a method for detecting nanoplastic content based on Py-GCMS technology of the present invention;
[0032] Figure 2 The SEM images of Escherichia coli (A) and protoplasts (B) of the present invention;
[0033] Figure 3 The extraction conditions of the present invention are optimized as follows: (A) BSA concentration; (B) pH value; (C) water bath temperature;
[0034] Figure 4 It is the H2O2 concentration diagram of the present invention;
[0035] Figure 5 The blank sample chromatogram and mass spectrum of the E. coli protoplasts (AB) of the present invention and the chromatogram and mass spectrum of the PS standard sample added (C–D);
[0036] Figure 6 The blank sample chromatogram and mass spectrum of the E. coli cell wall (AB) of the present invention and the chromatogram and mass spectrum of the PS standard sample (CD) added;
[0037] Figure 7 The growth curve of Escherichia coli of the present invention (A); the mass of nanoplastics in the cell wall and protoplasts (B);
[0038] Figure 8 The graph (AB) is a graph showing the recovery rates of nanoplastics with different physical and chemical properties of the present invention;
[0039] Fig. 9 It is a performance evaluation table of the method of the present invention. DETAILED DESCRIPTION
[0040] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0041] Embodiment 1:
[0042] See also Figures 1 to 9 As shown, the extraction and analysis of nanoplastics in cell wall solution includes the following steps:
[0043] S1. The method for expanding the culture of Escherichia coli from a monoclonal colony in step S1 is as follows: a monoclonal Escherichia coli colony is picked on LB solid agar medium and inoculated into 5 mL of LB liquid medium, the inoculated medium is cultured at 37° C. and 200 rpm overnight, and the bacterial solution is diluted with the medium to an OD of 600 The diluted bacterial solution was transferred to a centrifuge tube and centrifuged at 4°C, 3000 g for 20 min. The supernatant was discarded, and the bacterial cell pellet was collected. The bacteria were washed twice with 10 mM Tris-HCl buffer at pH 8.0 to remove the residual culture medium.
[0044] S2, separation of cell wall and protoplast, suspending the washed bacteria in Tris-HCl buffer, adding EDTA to a final concentration of 10 mM, shaking at 37°C and 100 rpm for 30 minutes to allow EDTA to complex with bacterial cell walls, 4000g, 4°C, 20 min, centrifuging again to remove the supernatant, adding 2 mg / mL lysozyme, continuing to shake at 37°C and 100 rpm for 30 minutes to further degrade the cell wall, and finally, separating the supernatant and the precipitate by centrifugation again;
[0045] S3, extraction of nanoplastics from the cell wall solution, taking 40 mL of the supernatant containing the cell wall in step S2, adding 1 μg of the synthesized nanoplastic, and adding 0.2 mL of 4 g / L BSA solution (i.e., the concentration of the BSA solution is 20 mg / L) thereto, and shaking at 300 rpm for 5 minutes to promote the binding of the nanoplastics with the BSA;
[0046] The solution pH was adjusted to 4.0 using diluted HNO3, and the solution was placed in a 60°C water bath for 10 minutes to allow the protein and nanoplastic to form a protein corona. After cooling, the solution was centrifuged at 8000 rpm for 10 minutes to precipitate the protein corona containing nanoplastic. The precipitate was redispersed in 200 μL of ultrapure water, transferred to an 80 μL pyrolysis sample cup, and dried at 100°C for 10 minutes.
[0047] Py-GC / MS was used for analysis. The parameters were set as follows:
[0048]
[0049]
[0050] The data is as follows:
[0051] Determination results: PS-NH2 spike concentration is 2.86ug / 4×10 10 CFU
[0052] The recovery rate of spiked product was 87%.
[0053] Embodiment 2:
[0054] See also Figures 1 to 9 As shown, extraction and analysis of nanoplastics in protoplasts:
[0055] Separate the cell wall and protoplasts according to the aforementioned method of step S1 and step S2 of Example 1;
[0056] Take 40 mL of the supernatant containing the cell wall in step S2, add 1 μg of the synthesized nanoplastic, and add 0.2 mL of 4 g / L BSA solution thereto, and shake at 300 rpm for 5 minutes to promote the binding of the nanoplastic with BSA;
[0057] The solution pH was adjusted to 4.0 using diluted HNO3, and the solution was placed in a 60°C water bath for 10 minutes to allow the protein and nanoplastic to form a protein corona. After cooling, the solution was centrifuged at 8000 rpm for 10 minutes to precipitate the protein corona containing nanoplastic. The precipitate was redispersed in 200 μL of ultrapure water, transferred to an 80 μL pyrolysis sample cup, and dried at 100°C for 10 minutes.
[0058] The method for extracting nanoplastics from the precipitate is as follows: 1 μg of the synthesized nanoplastic is added to the precipitate containing the protoplast sample in step S2, and then 200 μL of 6%, 12%, 18%, 24% and 30% H2O2 solutions are added to achieve the digestion of the protoplasts and release the nanoplastics therein, and the treated solution is transferred to a thermal pyrolysis sample cup for thermal pyrolysis and quantitative analysis of the nanoplastics;
[0059] Py-GC / MS was used for analysis, and the parameters were the same as those in Example 1.
[0060] Test results: The amount of nanoplastic spiked in the five groups of samples was 2.86ug / 4×10 10 CFU.
[0061] Recovery rate: The spiked recoveries were 67%, 70%, 86%, 83% and 84%, respectively.
[0062] Embodiment three:
[0063] See also Figures 1 to 9 As shown, the effect of nanoplastics on the growth of E. coli:
[0064] Separate the cell wall and protoplasts according to the aforementioned method of step S1 and step S2 of Example 1;
[0065] Extraction of nanoplastics from the cell wall solution: 40 mL of the supernatant containing the cell wall in step S2 was taken, 1 μg of the synthesized nanoplastics was added, and 0.2 mL of 4 g / L BSA solution was added thereto, and the mixture was shaken at 300 rpm for 5 minutes to promote the binding of the nanoplastics with the BSA;
[0066] The solution pH was adjusted to 4.0 using diluted HNO3, and the solution was placed in a 60°C water bath for 10 minutes to allow the protein and nanoplastic to form a protein corona. After cooling, the solution was centrifuged at 8000 rpm for 10 minutes to precipitate the protein corona containing nanoplastic. The precipitate was redispersed in 200 μL of ultrapure water, transferred to an 80 μL pyrolysis sample cup, and dried at 100°C for 10 minutes.
[0067] Extraction of nanoplastics in protoplasts: add 1 μg of synthesized nanoplastics to the precipitate containing the protoplast sample in step S2, and then add 200 μL of 18% H2O2 solution to achieve the digestion of the protoplasts and release the nanoplastics therein. Transfer the treated solution to a pyrolysis sample cup and heat it in a muffle furnace at 190°C for 3 hours to remove the interference of bacterial components and prepare for the quantitative analysis of nanoplastics.
[0068] like Figure 3 As shown, sample analysis and quantification, using Py-GC / MS for quantitative analysis of nanoplastics, the working parameters are optimized according to the experimental requirements, including pH value, BSA concentration and temperature, where the pH range is 2.5-4.5, the BSA concentration is 0-80 mg / L, and the temperature is 40-80 ° C. Three to five repeated experiments are performed on each sample to determine the spike recovery rate to ensure the accuracy and repeatability of the experimental results;
[0069] Effect of nanoplastics on the growth of Escherichia coli. PS-SO3H-NPs and PS-NH2-NPs were added to LB medium, and a blank control group was set up. The culture was carried out according to the above method and the OD was measured. 600 , determining the effects of different nanoplastics on the growth of Escherichia coli.
[0070] Specifically, the expression of the spike recovery rate in step S5 is:
[0071]
[0072] Among them, C e , unit μg / L, is the concentration of nanoplastics measured in a sample with a known added nanoplastic concentration of C, unit μg / L, while C0, unit μg / L, is the concentration of nanoplastics detected in a sample without added nanoplastics.
[0073] Experimental group: PS-SO3H-NPs and PS-NH2-NPs were added to LB medium, with the final concentration of 100 μg / L.
[0074] Control group: set up a blank control group;
[0075] Cultivation: Cultivate at 37°C, 200 rpm for 24 hours.
[0076] Determination: Determine OD 600 value
[0077] Py-GC / MS was used for analysis, and the parameters were the same as those in Example 1.
[0078] Data results:
[0079] PS-SO3H-NPs group: OD 600=1.00 (compared to the control group OD 600 =0.99, no effect on growth).
[0080] PS-NH2-NPs group: OD 600 =1.02 μg (no effect on growth).
[0081] From the above, we can see that the Py-GC / MS technology can accurately determine the content of nanoplastics in bacterial cell walls and protoplasts, reducing measurement errors. This method has high sensitivity and can detect low concentrations of nanoplastics. It is suitable for trace analysis and can evaluate the distribution and concentration of nanoplastics in bacteria. It can also provide an in-depth understanding of the effects of nanoplastics on microbial physiology and ecology. Through the study of different types of nanoplastics, its potential risks to microbial communities and ecosystems can be predicted.
[0082] By measuring the inhibitory effect of nanoplastics on the growth of Escherichia coli, we can understand the inhibitory mechanism of nanoplastics on microbial growth and compare the effects of different types of nanoplastics on bacterial growth, which can help select and design environmental protection measures. By accurately monitoring the ecological effects of nanoplastic pollution, we can provide data support for the formulation and implementation of more effective environmental protection measures.
[0083] Embodiment 4:
[0084] This design is specifically applied in the field of environmental protection. With the rapid development of nanotechnology, nanoplastics have gradually entered the ecological environment as emerging pollutants. Their tiny size and complex surface properties make it difficult for traditional detection methods to effectively deal with them. The new method based on Py-GC / MS can effectively solve this problem and achieve accurate quantification of nanoplastics in environmental media such as water, soil, and air, providing data support for environmental protection departments to formulate scientific pollution control strategies.
[0085] Further, the quantitative experiment of E. coli adsorption and ingestion of nanoplastics:
[0086] See also Figures 1 to 9 As shown, the cell wall and protoplasts were separated according to the aforementioned method of step S1 and step S2 of Example 1, and the nanoplastics were quantitatively detected. Escherichia coli was inoculated into 40 mL of LB medium at a ratio of 1:100, and the cells were collected by centrifugation at 4000 rpm for 10 min after culturing for 12 h. Subsequently, the bacterial cells were washed twice with PBS to remove the residues in the culture medium. After washing, the cells were resuspended in 40 mL of PBS, and 5 μg of the synthesized nanoplastic was added to the cell suspension, and the mixture was shaken at 37°C and 200 rpm for 6 h to promote the interaction between the nanoplastic and Escherichia coli. The effectiveness of the method was verified according to the experimental results, and optimized according to different experimental conditions.
[0087] Py-GC / MS was used for analysis, and the parameters were the same as those in Example 1.
[0088] Data results:
[0089] Nanoplastic content in the cell wall: PS-NH2 3.19±0.58μg, PS-SO3H 1.85±0.22.
[0090] Nanoplastic content in protoplasts: PS-NH2 0.52±0.03μg, PS-SO3H 0.48±0.01.
[0091] As can be seen from the above, the novel method based on Py-GC / MS technology of the present invention is used to quantitatively determine nanoplastics in Escherichia coli. Chemical reagents such as Tris-HCl, EDTA and lysozyme are used to effectively separate the cell wall and protoplasts of Escherichia coli, and Py-GC / MS analysis is performed under optimized experimental conditions, which can accurately determine the content of nanoplastics in these samples. In addition, the effectiveness of this method is verified by exposing newly cultured Escherichia coli in a phosphate buffer solution (PBS) containing nanoplastics.
[0092] The nanoplastics synthesized in the present invention have little effect on the growth of E. coli, and also reveal the different absorption characteristics of E. coli cell walls and protoplasts to nanoplastics. In particular, positively charged PS-NH2-NPs are easier to attach to the E. coli cell walls than negatively charged PS-SO3H-NPs, which indicates that it is related to the charge characteristics of E. coli itself. In addition, the key factors affecting the extraction efficiency of nanoplastics were systematically optimized, including BSA concentration, sample pH value and water bath temperature, to ensure the effective formation of the protein corona and the stable extraction of nanoplastics;
[0093] The present invention provides a new tool and method to monitor and evaluate the ecological effects of nanoplastics in environmental samples, and provides new insights into the potential impact of nanoplastics on microbial communities. The method demonstrates high accuracy, stability and repeatability, providing a scientific basis for future environmental monitoring. It is of great significance for a deep understanding of the behavior of nanoplastics in the environment and their impact on microorganisms, and also provides a new analytical method for the field of environmental science.
[0094] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. A method for detecting nanoplastic content based on Py-GCMS technology, characterized in that: include: S1. Expand the culture of E. coli from a single clone; S2, separating the cell wall of the Escherichia coli obtained in step S1, and centrifuging to obtain a supernatant and a precipitate; S3, extracting nanoplastics from the supernatant obtained in step S2; S4, extracting nanoplastics from the precipitate obtained in step S2; S5. Use Py-GC / MS to quantitatively analyze the nanoplastics in steps S3 and S4; S6. Analyze the effect of nanoplastics on Escherichia coli; S7. Conduct a quantitative experiment on the adsorption and uptake of nanoplastics by Escherichia coli and optimize it according to different experimental conditions.
2. The method for detecting nanoplastic content based on Py-GCMS technology according to claim 1, characterized in that: The method for expanding the culture of Escherichia coli from a monoclonal colony in step S1 is as follows: a monoclonal Escherichia coli colony is picked from the LB solid agar medium and inoculated into 5 mL of LB liquid medium, the inoculated medium is cultured at 37°C and 200 rpm overnight, and the bacterial solution is diluted with the medium to an OD of 600 The diluted bacterial solution was transferred to a centrifuge tube and centrifuged at 4°C, 3000g for 20 min. The supernatant was discarded, the bacterial cell pellet was collected, and the bacteria were washed twice with 10mM Tris-HCl buffer, pH 8.0, to remove the residual culture medium.
3. The method for detecting nanoplastic content based on Py-GCMS technology according to claim 1, characterized in that: In step S2, the cell wall of Escherichia coli is separated, and the washed bacteria are suspended in Tris-HCl buffer with a pH of 8.0, EDTA is added to a final concentration of 10 mM, and the mixture is shaken at 37° C. and 200 rpm for 30 minutes to allow EDTA to complex with the bacterial cell wall. The mixture is centrifuged at 4000 g and 4° C. for 20 minutes to remove the supernatant, 2 mg / mL of lysozyme is added, and the mixture is shaken at 37° C. and 200 rpm for 60 minutes to further degrade the cell wall. Finally, the mixture is centrifuged again to obtain a supernatant and a precipitate.
4. The method for detecting nanoplastic content based on Py-GCMS technology according to claim 3, characterized in that: The supernatant is a cell wall component, and the precipitate is Escherichia coli without a cell wall, namely, protoplasts.
5. The method for detecting nanoplastic content based on Py-GCMS technology according to claim 1, characterized in that: The method for extracting nanoplastics from the supernatant in step S3 is as follows: take 40 mL of the supernatant containing cell walls in step S2, add 1 μg of synthesized nanoplastics, and add 0.2 mL of 4 g / L BSA solution thereto, use an orbital shaker, and oscillate at 300 rpm for 5 minutes to promote the binding of nanoplastics and BSA to obtain a binding solution; use diluted HNO3 to adjust the pH of the binding solution to 4.0, heat it in a 60°C water bath for 10 minutes, so that the protein and the nanoplastic form a protein corona, after cooling, centrifuge at 8000 rpm for 10 minutes to precipitate the protein corona containing nanoplastics, and redisperse the protein corona in 200 μL of ultrapure water, transfer it to an 80 μL thermal lysis sample cup, and dry it at 100°C for 10 minutes.
6. The method for detecting nanoplastic content based on Py-GCMS technology according to claim 1, characterized in that: The method for extracting nanoplastics from the precipitate in step S4 is as follows: 1 μg of the synthesized nanoplastic is added to the precipitate containing the protoplast sample in step S2, and then 200 μL of H2O2 solutions of different concentrations are added to achieve the digestion of the protoplasts and release the nanoplastics therein, and the treated solution is transferred to a thermal pyrolysis sample cup for thermal pyrolysis and quantitative analysis of the nanoplastics.
7. The method for detecting nanoplastic content based on Py-GCMS technology according to claim 1, characterized in that: The quantitative analysis method in step S5 is: using Py-GC / MS for quantitative analysis of nanoplastics, wherein the working parameters include pH value, BSA concentration and temperature, which are optimized according to the experimental requirements, and each sample is repeated three to five times to determine the spike recovery rate.
8. The method for detecting nanoplastic content based on Py-GCMS technology according to claim 7, characterized in that: The expression of the spike recovery is: Among them, C e , unit μg / L, is the concentration of nanoplastics measured in a sample with a known added nanoplastic concentration of C, unit μg / L, while C0, unit μg / L, is the concentration of nanoplastics detected in a sample without added nanoplastics.
9. The method for detecting nanoplastic content based on Py-GCMS technology according to claim 1, characterized in that: Step S6: The method for analyzing the effect of nanoplastics on Escherichia coli is as follows: PS-SO3H-NPs and PS-NH2-NPs are added to LB medium, a blank control group is set up, and the culture is carried out according to the above method and the OD is measured. 600 , determining the effects of different nanoplastics on the growth of Escherichia coli.
10. The method for detecting nanoplastic content based on Py-GCMS technology according to claim 1, characterized in that: The method of the quantitative experiment in step S7 is to inoculate Escherichia coli into 40 mL of LB medium at a ratio of 1:100, and after culturing for 12 hours, centrifuge at 4000 rpm for 10 minutes to collect the cells. Subsequently, the bacterial cells are washed twice with PBS to remove the residues in the culture medium. After washing, the cells are resuspended in 50 mL of PBS, and 5 μg of synthetic nanoplastic is added to the cell suspension, and oscillated at 37°C and 200 rpm for 6 hours to promote the interaction between the nanoplastic and Escherichia coli.