CN112229921A - Detection method for nano-plastic in biological tissue - Google Patents
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Abstract
The invention discloses a method for detecting nano-plastic in biological tissues, and belongs to the field of analysis of nano-plastic. The method comprises the following steps: (1) digesting aquatic animal tissues by an alkali digestion method; (2) precipitating the protein with the extracted nano-plastic in the digestion solution obtained in the step (1) by adopting a protein precipitation method; (3) and (3) carrying out quantitative analysis on the precipitate obtained in the step (2) by adopting pyrolysis gas chromatography-mass spectrometry. The invention realizes the component identification and quality quantification of the nano-plastic in the complex biological tissue. Under the conditions of optimized alkali types, concentrations and the like, the detection limits of the method for the Polystyrene (PS) nano plastic and the polymethyl methacrylate (PMMA) nano plastic are 0.03 mu g/g and 0.09 mu g/g respectively. The method also maintains the original shape and size of the nano plastic, has the advantages of good reproducibility and high sensitivity, and can be used for quantitative analysis of various nano plastics.
Description
Technical Field
The invention belongs to the field of analysis of nano-plastics, and particularly relates to a method for detecting nano-plastics in biological tissues.
Background
Micro-plastics (<5mm) and nano-plastics (<1um) have been recognized as ubiquitous environmental pollutants (rillg, m.c.; Lehmann, a., et al, Science 2020,368, 1430-1431). There is increasing evidence that micro-and nano-plastics are taken up by animals, distributed within the animal body, accumulated in various tissues, and transported through the trophic chain. Thus, micro-and nano-plastics in the body have become threats to living and human health. However, research related to micro-and nano-plastics in biological systems has been hampered by the lack of methods to characterize and quantify these particles in complex biological matrices. Currently, the main methods used for microplasticity studies are visual inspection, fourier transform infrared spectroscopy (FTIR) and raman spectroscopy. However, these methods are generally limited to particles of 5mm to 20um, and few reports have focused on the fraction below 20 um. Due to the limitation of spatial resolution, common micro-plastic optical detection technologies, such as micro-imaging technologies based on infrared spectroscopy and raman spectroscopy, are often difficult to be directly applied to the detection of nano-plastics. Furthermore, the results obtained by these techniques are not decisive for external factors (such as the presence of organic matter), and lack of quantitative analysis, thus causing errors and omissions. Thermal cracking combined with gas chromatography-mass spectrometry (Py-GC/MS) is a promising approach. Although this technique can reliably identify and accurately quantify micro-nano plastics without being limited by particle size, it is not easy to extract and measure micro-nano plastics from complex biological matrices. One study analyzed by Py-GC/MS for greater than 2.7 μm of microplastics in australian seafood (Ribeiro, f.; okoflo, e.d.; et al, environ.sci.technol.2020), while no investigation has been reported for nanoplastic contamination in animals due to the lack of suitable extraction and analysis methods. Alkaline digestion with sodium hydroxide (NaOH), potassium hydroxide (KOH) is commonly used to release micro-plastics and inorganic nanoparticles from tissues, but they have not been applied to nano-plastics. Therefore, it is of great significance to develop a method for extracting and analyzing nano-plastics from biological tissues. There is no research report on the method for extracting and analyzing nano plastic from biological tissues.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a detection method for nano-plastic in biological tissues.
The purpose of the invention is realized by the following technical scheme.
A method for detecting nanoplastic in biological tissue, comprising the steps of:
(1) digesting animal tissues by an alkali digestion method;
(2) precipitating the protein with the extracted nano-plastic in the digestion solution obtained in the step (1) by adopting a protein precipitation method;
(3) and (3) carrying out quantitative analysis on the precipitate obtained in the step (2) by using pyrolysis gas chromatography-mass spectrometry (Py-GC/MS).
Preferably, the base in step (1) is a NaOH (sodium hydroxide) solution or a TMAH (tetramethylammonium hydroxide) solution.
Preferably, the alkali used for alkali digestion is tetramethylammonium hydroxide solution, and the highest recovery rate and the lowest substrate interference are obtained by digesting the biological tissue with TMAH alkali.
Preferably, the concentration of the sodium hydroxide solution is 2.5-15M.
Preferably, the concentration of the sodium hydroxide solution is 10M.
Preferably, the concentration of the tetramethylammonium hydroxide solution is 5 wt% to 25 wt%.
Preferably, the concentration of the tetramethylammonium hydroxide solution is 15 wt%.
Preferably, the protein precipitation method of step (2) comprises the steps of: filtering the digestion solution obtained in the step (1) by using a glass fiber filter membrane, adding ethanol into the filtrate, incubating in a water bath at the temperature of 60-80 ℃, gradually generating flocculent protein in the solution, and then centrifuging to ensure that the protein is completely precipitated.
Preferably, the temperature of the water bath is 80 ℃.
Preferably, the glass fiber filter has a pore size of 1um and a diameter of 25mm, and is purchased from Whatman.
Preferably, the centrifugation rate is 1500-.
Preferably, in the step (3), the precipitate obtained in the step (2) is dispersed by water and then dried, and then the obtained dispersion liquid is subjected to quantitative analysis by adopting a pyrolysis gas chromatography-mass spectrometry method; the concentration of the precipitate in the dispersion liquid is 10ug/L-10 mg/L; the drying temperature is 100 deg.C, and the drying time is 10 min.
Preferably, the animal tissue is aquatic animal tissue.
Preferably, the nano plastic comprises more than one of polystyrene, polymethyl methacrylate, polyethylene, polyvinyl chloride and polyethylene terephthalate nano plastic.
Preferably, the particle size of the nano plastic is 50nm-100 nm.
Preferably, in the cracking gas chromatography-mass spectrometry of the step (3), styrene trimer (m/z91) is selected as an indicator of polystyrene in the nanoplastic, and methyl methacrylate (m/z100) is selected as an indicator of polymethyl methacrylate in the nanoplastic.
The invention adopts unique alkali digestion and protein extraction processes to realize component identification and quality quantification of the nano-plastic in the complex biological tissue through Py-GC/MS analysis for the first time. Wherein, the biological tissue is digested by alkali to obtain higher recovery rate and lower matrix interference, the nano plastic in the biological tissue sample is extracted by a protein precipitation method, and a Py-GC/MS spectrum analysis is adopted to obtain a specific cracking product of the nano plastic so as to quantitatively analyze the nano plastic. The method has low detection limit (0.15 amol/g for 100nm PS nano plastic and 0.40amol/g for 100nm PMMA nano plastic), and the size and the shape of the extracted nano plastic are maintained, so that the extracted nano plastic can be further characterized. The method is used for directly identifying the composition of the nano-plastics in the aquatic animal tissues and quantifying the composition, so that the method is helpful for filling up the necessary blank of metrology, and further clarifying the potential fate and toxicity of the nano-plastics.
Compared with the prior art, the invention has the following advantages and technical effects:
(1) the invention realizes the component identification and quality quantification of the nano-plastic in the complex biological tissue for the first time.
(2) The detection limit of the method for the polystyrene nano plastic and the polymethyl methacrylate nano plastic respectively reaches 0.03 mu g/g and 0.09 mu g/g.
(3) The invention adopts TMAH alkali to digest biological tissues to obtain the highest recovery rate and the lowest matrix interference.
(4) The invention keeps the size and shape of the nano plastic in the extraction process, thereby being capable of carrying out various researches on the original physicochemical property of the nano plastic.
(5) The method is simple and effective, and has the characteristics of high specificity, high repeatability and low detection limit.
Drawings
FIG. 1 shows Py-GC/MS chromatograms of digested, protein-extracted fish samples of NaOH (A) and TMAH (B) at different concentrations in example 1.
FIG. 2 is a graph showing the effect of the type and concentration of the base on the recovery rate of PS nanoplastic in example 2.
FIG. 3 is TEM image and size distribution graph of PS nanoplastic before and after (A) extraction from minced fish meat tissue in example 3.
FIG. 4 is a schematic flow chart of the method for quantitatively analyzing nanoplastic in animal tissue according to example 6.
FIG. 5 is a Py-GC/MS spectrum of a sample obtained by subjecting the aquatic animal tissue (A, B is loach, C, D is Eriocheir sinensis) to alkali digestion and protein precipitation in example 6.
Detailed Description
Specific embodiments of the present invention will be described in further detail below with reference to examples and drawings, but the present invention is not limited thereto.
The parameters for the Py-GC/MS analysis in each example are as follows:
example 1
Adding 1mL of 15M NaOH or 25% wt TMAH into 0.5g of minced fish sample without nanoplastic, and performing alkaline digestion to obtain 50mu.L of the resulting solution was dried at 100 ℃ for 20min in a thermal cracking sample cup and then subjected to Py-GC/MS measurement. Meanwhile, after 0.5g of minced fish sample without nanoplastic is respectively added with 1mL of 15M NaOH or 25% wt TMAH for alkali digestion, the digestion solution is filtered by a glass fiber filter membrane (with the aperture of 1um and the diameter of 25mm), 10mL of ethanol (95%) is added into the filtrate, water bath at 80 ℃ is carried out for 30min, then the mixture is centrifuged at 2000rpm for 5min, supernatant is removed, and 0.5mL of H is added into protein precipitate2O is dispersed, 50 mu L of the dispersion liquid is taken out to be arranged in a thermal cracking sample cup, dried for 20min at 100 ℃ and then subjected to Py-GC/MS measurement.
As can be seen from A, B in fig. 1, the number of peaks was significantly reduced after protein extraction, and the signal intensity of NaOH and TMAH was significantly reduced with increasing alkali concentration. Especially, when TMAH is used for digestion, the number of main peaks is much smaller, which indicates that most of sample matrixes can be eliminated by extracting protein after TMAH digestion.
Example 2
Adding 2 μ g/g PS nano plastic into 0.5g minced fish sample without nano plastic, performing alkaline digestion with 1mL NaOH or TMAH of various concentrations, filtering digestion solution with glass fiber filter membrane (aperture of 1um, diameter of 25mm), adding 10mL ethanol (95%) into filtrate, performing 80 deg.C water bath for 30min, centrifuging at 2000rpm for 5min, removing supernatant, adding 0.5mL H into protein precipitate2O is dispersed, 50 mu L of the dispersion liquid is taken out to be arranged in a thermal cracking sample cup, dried for 20min at 100 ℃ and then subjected to Py-GC/MS measurement.
As can be seen from FIG. 2, the recovery rate of the nanoplastic reaches the maximum when the concentrations of NaOH and TMAH are 10M and 15% (w/w), respectively. Further increase in alkali concentration did not increase the recovery efficiency. Therefore, the alkaline digestion with 15% (w/w) TMAH has the best effect due to the guarantee of the highest recovery rate of the nano plastic and the weak interference of the matrix.
Example 3
Adding 50 μ g/g PS nano plastic into 0.5g minced fish sample without nano plastic, performing alkaline digestion with 1mL 25% wt TMAH, filtering digestion solution with glass fiber filter membrane (aperture of 1um, diameter of 25mm), adding 10mL ethanol (95%) into filtrate, performing 80 deg.C water bath for 30min, centrifuging at 2000rpm for 5min, removing supernatant, and adding egg0.5mL of H was added to the white precipitate2O, dispersing, dripping 5 mu L of the dispersed solution on a copper net, and drying for 2 hours in vacuum at 40 ℃. Meanwhile, 5 mu LPS nano plastic solution is dropped on a copper net and dried for 2h in vacuum at 40 ℃. And observing the shapes of the nano-plastics in the two samples by adopting a Transmission Electron Microscope (TEM), and carrying out particle size statistics on the PS nano-plastics in the TEM image by using Nanosizer software to obtain the particle size distribution of the PS nano-plastics before and after the extraction of the samples.
As can be seen from FIG. 3, the particle size distribution of the PS nanoplastic after extraction is (94.5. + -. 5.3) nm, which is close to (98.1. + -. 5.9) nm before extraction. Meanwhile, no significant shape change occurred after extraction. The method has no obvious influence on the shape and the particle size of the nano plastic.
Example 4
Adding 1mL of 25 wt% TMAH into 0.5g of minced aquatic animal tissue, performing alkaline digestion, filtering with glass fiber filter membrane (aperture of 1um, diameter of 25mm), adding 10mL of ethanol (95%) into the filtrate, performing 80 deg.C water bath for 30min, centrifuging at 2000rpm for 5min, removing supernatant, adding 0.5mL of H into protein precipitate2O is dispersed, 50 mu L of the dispersion liquid is taken out to be arranged in a thermal cracking sample cup, dried for 20min at 100 ℃ and then subjected to Py-GC/MS measurement. Simultaneously, selecting an aquatic animal tissue, adding 2.12 mu g/g PS nano plastic and 1.84 mu g/g PMMA nano plastic, adding 1mL of 25wt TMAH for alkali digestion, adding 10mL of ethanol, carrying out water bath at 80 ℃ for 30min, centrifuging at 2000rpm for 5min, removing supernatant, adding 0.5mL of H into protein precipitate2O is dispersed, 50 mu L of the dispersion liquid is taken out to be arranged in a thermal cracking sample cup, the drying is carried out for 20min at the temperature of 100 ℃, and the Py-GC/MS measurement is carried out.
In some embodiments of the invention, the nanoplastic comprises polystyrene nanoplastic (PS) and polymethylmethacrylate nanoplastic (PMMA) and the selected particle sizes are 50nm and 100 nm. The experiment investigates the influence of different types and particle sizes of nano-plastics on the extraction, and the result has no obvious difference. The method is suitable for extracting various kinds of nano plastics in biological tissues.
Example 5
0.5. mu.g/g of poly in 0.5g of minced, respectively nanoplastic-free fish samplesAdding 1mL of 15M NaOH or 25 wt% of TMAH into styrene nano plastic and polymethyl methacrylate nano plastic for alkali digestion, filtering the digestion solution with a glass fiber filter membrane (the aperture is 1um and the diameter is 25mm), adding 10mL of ethanol into the filtrate, carrying out water bath at 80 ℃ for 30min, centrifuging at 2000rpm for 5min, removing the supernatant, adding 0.5mL of H into the protein precipitate2O is dispersed, 50 mu L of the dispersion liquid is taken out to be arranged in a thermal cracking sample cup, dried for 20min at 100 ℃ and then subjected to Py-GC/MS measurement. Obtaining signals of the polystyrene nano plastic and the polymethyl methacrylate nano plastic corresponding to 3 times of signal-to-noise ratio, namely obtaining detection limits. The detection limits of the polystyrene nano plastic and the polymethyl methacrylate nano plastic by adopting the method of the embodiment respectively reach 0.03 mu g/g and 0.09 mu g/g.
Example 6: quantitative analysis of nanoplastic in tissue of 14 aquatic animals
(1) Extraction of nano-plastics from biological tissue
0.5g of 14 (as shown in the table below) minced aquatic animal tissues were placed in 15ml centrifuge tubes, 1ml of TMAH (15%, w/w) was added separately and shaken at 300rpm for 24h at room temperature until the tissues were completely digested. The digestion solution was then filtered through a glass fiber filter (pore size 1um, diameter 25mm), and 10mL of ethanol (95%) was added to the filtrate and incubated in a water bath at 80 ℃ for 30 min. During this time, flocculent protein gradually appeared in the solution, which was then centrifuged at 2000rpm for 5min to completely precipitate the protein. After removing the supernatant, 0.5mLH was added2O, redispersing the protein with extracted nanoplastic.
(2) Py-GC/MS analysis
50 μ L of the nanoplastic-containing dispersion was transferred to an 80 μ L pyrolysis target cup and dried at 100 ℃ for 10min to remove water for subsequent Py-GC/MS measurement, and the flow diagram is shown in FIG. 4. Since organic matter, such as chitin and proteins, is certainly present in aquatic animals, styrene (m/z 104) is released during pyrolysis, and although 104 styrene is an abundant pyrolysis product of PS, it is not suitable for identification and quantification of PS in tissues. In contrast, styrene trimer (m/z 312) is a polymer-specific compound of PS, and the ion-rich fragment m/z91 of styrene trimer was chosen as an indicator for PS quantitation. For PMMA nano plastic, methyl methacrylate (m/z100) with the most abundant content and the strongest polymer specificity is selected as an indicator. And finally, the type and the content of the nano plastic can be measured according to a Py-GC/MS spectrum. As shown in fig. 5, after alkali digestion and protein extraction, m/z10, 91 and 312 peaks are extracted from loach (a in fig. 5) and crab larvae (B in fig. 5), the retention time is consistent with that of a PS standard, and meanwhile, the extracted peaks can be obtained as styrene and styrene trimer signals through mass spectrograms (C and D in fig. 5), which further proves the existence of PS nanoplastic in a biological sample.
Claims (10)
1. A method for detecting nanoplastic in biological tissue, comprising the steps of:
(1) digesting animal tissues by an alkali digestion method;
(2) precipitating the protein with the extracted nano-plastic in the digestion solution obtained in the step (1) by adopting a protein precipitation method;
(3) and (3) carrying out quantitative analysis on the precipitate obtained in the step (2) by adopting pyrolysis gas chromatography-mass spectrometry.
2. The method for detecting nanoplastic in biological tissue as claimed in claim 1, wherein the alkali used in the alkali digestion in step (1) is sodium hydroxide solution or tetramethylammonium hydroxide solution.
3. The method for detecting nanoplastic in biological tissue according to claim 2, wherein the concentration of the sodium hydroxide solution is 2.5-15M; the concentration of the tetramethylammonium hydroxide solution is 5-25 wt%.
4. The method for detecting nanoplastic in biological tissue according to claim 3, wherein the concentration of the sodium hydroxide solution is 10M; the concentration of the tetramethylammonium hydroxide solution is 15 wt%.
5. The method for detecting nanoplastic in biological tissue according to claim 1, wherein the protein precipitation method in step (2) comprises the following steps: filtering the digestion solution obtained in the step (1) by using a glass fiber filter membrane, adding ethanol into the filtrate, incubating in a water bath at the temperature of 60-80 ℃, gradually generating flocculent protein in the solution, and then centrifuging to ensure that the protein is completely precipitated.
6. The method as claimed in claim 5, wherein the glass fiber filter membrane has a pore size of 1um and a diameter of 25 mm.
7. The method as claimed in claim 5, wherein the centrifugation speed is 1500-.
8. The method for detecting nanoplastic in biological tissue as claimed in claim 1, wherein in step (3), the precipitate obtained in step (2) is dispersed with water, then dried, and then the dispersion obtained is subjected to quantitative analysis by pyrolysis gas chromatography-mass spectrometry; the concentration of the precipitate in the dispersion is 10ug/L-10 mg/L.
9. The method as claimed in claim 1, wherein the nanoplastic comprises one or more of polystyrene, polymethyl methacrylate, polyethylene, polyvinyl chloride and polyethylene terephthalate nanoplastic; the animal tissue is aquatic animal tissue.
10. The method for detecting nanoplastic in biological tissue as claimed in any one of claims 1 to 9, wherein in the pyrolysis gas chromatography-mass spectrometry of step (3), styrene trimer is selected as an indicator of polystyrene in the nanoplastic, and methyl methacrylate is selected as an indicator of polymethyl methacrylate in the nanoplastic.
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