CN108181154B - Method for detecting micro-plastics in organism - Google Patents
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Abstract
The invention relates to a method for detecting micro-plastics in a biological sample, which comprises the following steps: s110, digestion: soaking the biological sample in 50-70% nitric acid at room temperature for 6-18 hr, heating to 60-95 deg.C and maintaining for 1-4 hr to obtain digested biological sample; s120, degreasing: washing and filtering the digested organism sample obtained in the step S110 with a surfactant solution at the temperature of 60-95 ℃ while the digested organism sample is hot to obtain degreased micro-plastic; and S130, washing and filtering the degreased micro plastic obtained in the step S120 by using water at the temperature of 60-95 ℃ to obtain the cleaned micro plastic. The method can quickly and accurately carry out qualitative and quantitative detection on the micro-plastic entering the organism, realizes the purpose of detecting the enrichment and distribution of the micro-plastic in the organism, and provides a practical tool for environmental monitoring.
Description
Technical Field
The invention belongs to the field of detection of environmental pollutants, and particularly relates to a method for detecting micro-plastics in organisms, in particular to a method for detecting the micro-plastics in the organisms by using a fluorescence microscope counting method.
Background
The plastic is used in daily life in large quantity due to the characteristics of low cost, stable chemical property, good impact resistance and the like, but the environmental pollution is aggravated because the plastic is difficult to degrade, and the marine plastic pollution condition is particularly prominent. Plastic pollutants in seawater are gradually degraded into tiny particles under the factors of light, heat, organisms and the like, and the tiny particles are called as micro plastics. Once the micro-plastic pollution in water is increased, the marine organism is subjected to toxicological reaction, such as induction of genetic aberration, reproduction abnormality, hormone disorder or toxicological change of tissues and the like. The research on the components and the content of the micro-plastic in the oysters can not only clarify the pollution mechanism, but also provide guarantee for the safety of the marine food.
Therefore, there is still a need to develop new methods for detecting microplastics in organisms.
Disclosure of Invention
In order to solve the above problems in the prior art, the present invention provides the following technical solutions.
One aspect of the present invention provides a method for detecting a micro-plastic in a sample of a biological body, comprising:
s100, a step of extracting the micro-plastic from the biological sample, which comprises:
s110, digestion: soaking a biological sample in 50-70% nitric acid at room temperature for 6-18 hours, heating to 60-95 ℃ and maintaining for 1-4 hours to obtain a digested biological sample;
s120, degreasing: washing and filtering the digested organism sample obtained in the step S110 with a surfactant solution at the temperature of 60-95 ℃ while the digested organism sample is hot to obtain degreased micro-plastic;
and S130, washing and filtering the degreased micro plastic obtained in the step S120 by using water at the temperature of 60-95 ℃ to obtain the cleaned micro plastic.
Further, step S100 further includes: and adding the obtained cleaned micro-plastic into water, and then carrying out ultrasonic treatment to obtain a micro-plastic suspension.
Further, the suction filtration is a suction filtration with a microporous filtration membrane.
Further, step S100 further includes:
s140, grading: grading the micro plastic according to the density by using solutions with different densities; preferably, the solution is selected from a density of 1g/cm 3 Has a density of 1.19g/cm 3 The saturated sodium chloride aqueous solution of (2) has a density of 0.91g/cm 3 58.4% ethanol aqueous solution with a density of 0.925g/cm 3 55.4% ethanol aqueous solution, and a density of 1.27g/cm 3 27.5% calcium chloride in water.
Further, the surfactant is sodium lauryl sulfate.
Further, the method for detecting the micro-plastic in the organism further comprises the following steps:
s200, dyeing the cleaned micro plastic obtained in the step S100, wherein the dyeing comprises the following steps: contacting the micro-plastic with aminosiloxane to obtain the micro-plastic with amino groups, and then contacting the micro-plastic with fluorescein isothiocyanate to obtain the dyed micro-plastic.
Further, in step S200, the aminosiloxane is (3-aminopropyl) trimethoxysilane, and methanol and (3-aminopropyl) trimethoxysilane are added to the microplastic in a ratio of 19.
Further, the method for detecting the micro-plastic in the organism further comprises the following steps:
s300, the dyed microplastic obtained in step S200 is observed with a microscope, and the microplastic particles are counted.
Further, the method for detecting the micro-plastic in the organism further comprises the following steps:
and S400, carrying out infrared spectrum detection or electron microscope characterization on the cleaned micro plastic obtained in the step S100.
The method comprises the steps of exploring and optimizing a micro-plastic extraction process, dyeing and carrying out fluorescence counting on the micro-plastic by using a silanization technology, and finally characterizing and analyzing the plastic types by using a Fourier infrared spectrum and an electron microscope (SEM). In particular, the method comprises the steps of extracting the micro-plastics from oysters, optimizing an extraction process, simultaneously dyeing the extracted micro-plastics by using a silanization technology and combining fluorescence, and finally observing the types and the morphological characteristics of the micro-plastics by means of Fourier infrared spectroscopy (FT-IR) and an electron microscope (SEM). The research proves that: the oysters in the Zhanjiang sea area do contain micro-plastics, and the best extraction process is to digest the oysters with 25mL concentrated nitric acid, and sodium dodecyl sulfate is used as a surfactant to remove fat; the optimal staining method is to modify amino groups on the surface of the micro plastic by using methanol and (3-aminopropyl) trimethoxy silane, wherein the amino groups are combined with Fluorescein Isothiocyanate (FITC) to generate fluorescence, and the content of the sample is 67368 per gram by counting through a fluorescence microscope. The microplastic samples may contain polypropylene (PP) or polyvinyl chloride (PVC) by electron microscopy (SEM) and fourier-infrared spectroscopy.
The method can quickly and accurately carry out qualitative and quantitative detection on the micro-plastic entering the organism, realizes the purpose of detecting the enrichment and distribution of the micro-plastic in the organism, and provides a practical tool for environmental monitoring.
Drawings
FIG. 1 is a bright field micrograph (4 x) of the microplastic in the oyster, where (a), (b) and (c) are images in different fields of view;
FIG. 2 is a bright field micrograph (4X) of a small piece of plastic cut out of a plastic bag in a pre-experiment for plastic staining;
FIG. 3 is a fluorescence micrograph (4 ×) of FITC-modified plastic in a 4-fold dark field of the microscope in a pre-experiment with staining of the plastic, wherein (a), (b) and (c) are all FITC-modified plastics;
FIG. 4 is a fluorescent micrograph (4 x) of the microplastic in oysters showing the morphology of the microplastic staining clearly observed in the microplastic staining preliminary experiment, wherein (a), (b) and (c) are all microplastics in oysters;
FIG. 5 is the results of a microplastic staining experiment, wherein (a), (b) and (c) are fluorescence counting micrographs (4X) of microplastic in oysters;
FIG. 6 is an infrared spectrum of the micro-plastic in oyster;
FIG. 7 is a graph of the infrared spectra of polypropylene (left) and polyvinyl chloride (right) of the prior art;
FIG. 8 is an electron microscopic image of the sample of the micro-plastic in oysters, wherein (a), (b) and (c) are all electron microscopic images of the micro-plastic in oysters.
Detailed Description
Exemplary embodiments of the invention are described and illustrated below. For clarity and accuracy, the exemplary embodiments discussed below may include preferred steps, methods and features that one of ordinary skill in the art would recognize are not necessarily required to fall within the scope of the invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1: detection of micro-plastics in oysters
1.1 pretreatment of oysters
(1) Taking out multiple oysters harvested from Zhanjiang sea area from a refrigerator, thawing, removing shells, and cleaning with water.
(2) The cleaned oysters were weighed on an electronic balance and the weights were recorded separately.
1.2 exploration of digestion Experimental conditions
The oyster is digested by the following methods: an enzyme digestion method, a nitric acid and 30% hydrogen peroxide digestion, a nitric acid digestion, a saturated sodium chloride layering method and an ethanol layering method.
1.2.1 enzymatic digestion
(1) After 5 oysters (114 g) were taken out from a refrigerator and pretreated according to 3.1 steps, the oysters were crushed into slurry by a juicer (JYL-C52V multifunctional juicer, jiuyang corporation, the same below), 500mL of water and 5g of papain (papain, qiyun biotechnology limited, guangzhou, the same below) were added, and the mixture was allowed to stand overnight to observe whether the oysters could be decomposed by the papain.
(2) Taking an oyster (11.52 g), pretreating according to the 3.1 steps, crushing into slurry by using a juicer, adding 50mL of water, boiling for 30min, adding 0.9g of papain, and standing overnight in a water bath kettle at 50 ℃.
The experiment is carried out according to the method of the experiment (1), and the solution still presents turbid green after standing overnight, and simultaneously, a blocky object can be observed, so that the method is preliminarily considered to be unsuccessful in digestion. Experiment (1) was optimized and improved by heating oysters to denature their proteins and remove most of the organics, while cooling to about 50 deg.C, adding papain, and standing overnight in a water bath at 50 deg.C to allow the papain to decompose the remaining organics. After overnight, the solution was slightly pink turbid, so 58% ethanol was added to allow it to separate, but the effect was still insignificant without a clear, transparent liquid layer.
1.2.2 nitric acid digestion method
The pretreated oysters were rinsed once with ultrapure water and placed in a kjeldahl flask for the experiments in table 1.
Table 1: nitric acid digestion series experiment
First, 1.2.2 experiments 1 and 2 were carried out using a combination of 18mL 69-HO3 +6mL 30% H2O2 with a ratio of 69% nitric acid to 30% hydrogen peroxide 3:1, with experiment 1 being heated for 10 minutes and experiment 2 being subjected to a 12-hour overnight standing method for comparative reference. It was found that the entire oyster was digested by the 10 minute heating in experiment 1, but there were a large amount of foam and grease-like substances, and some fine particles precipitated on the bottom, and the preliminary judgment was fine sand, and therefore, a large amount of foam and grease-like substances could basically judge that the experiment failed to be completely digested; after standing for 12 hours in experiment 2, the whole oyster still could not be digested, and then the oyster was heated for 5 minutes to be digested into the state of experiment 1.
In summary, it is not determined whether the digestion solution is effective at present, but it is certain whether the heating and the length of the heating time will affect the digestion degree, so that the heating is kept and the heating time is controlled when the next experiment is carried out, and meanwhile, the standing time is changed to explore the influence degree of the standing time on the digestion.
Next, 69% nitric acid was used directly as a digestion solution and left to stand overnight before heating, heated to a boiling state and held for 2 hours, whereupon a series of experiments 3,4,5,6 in table 1 were designed. The results are shown in Table 2.
Table 2: results of a series of experiments on nitric acid digestion
The result can find that a large amount of large-block granular substances exist in experiment 3, the large-block granular substances float on the upper layer of the digestion solution in a centralized manner, the liquid is in a light yellow shape, the fat is preliminarily judged to be coagulated into a block shape and is not beneficial to next extraction, meanwhile, experiments 5 and 6 have the same condition, the fat is probably condensed into a block due to too long standing digestion time, only experiment 4 is carried out, and the grease on the surface is in a liquid shape, so that the conditions of experiment 3, experiment 5 and experiment 6 can be preliminarily judged to be infeasible, the reason is that a large amount of fat solids are not beneficial to next extraction, the micro plastic contained in the solid fat can be removed together if the fat is directly removed, the fat in experiment 4 is in a liquid shape, the fat only needs to be removed together with water, the most feasible method at present is to use a suction filtration device, a filter membrane with a proper pore diameter is selected, the micro plastic can be left on the filter membrane, and other impurities are filtered together with the water.
1.2.3 degreasing experiments
The pretreated oysters were taken, rinsed with ultrapure water, placed in a kjeldahl flask, and after treatment according to experiment 4 of 1.2.2, experiments 9 and 10 were performed according to the following protocol, wherein experiments 7 and 8 are preliminary experiments for degreasing and serve as a reference.
TABLE 3 degreasing experiments
Through experiment 4 of 1.2.2, the digestion solution is known to have a large amount of liquid grease on the surface, and a 0.22-micron microporous filter membrane (Shanghai Xinghai Sundai clean materials factory) is selected for suction filtration, so that the liquid grease can not permeate the filter membrane. The pore diameter of the micro-plastic researched by the experiment is less than 5 mm. Therefore, experiment 9 scheme was carried out, through adding 1mol NaOH at the suction filtration in-process, made grease and alkali take place saponification to wash with a large amount of ultrapure water, thereby eliminated the influence of grease, but the practical effect is that grease and alkali take place saponification, can not make grease become the micromolecule and filtered, and the filter membrane surface still has a large amount of grease, blocks up the suction filtration device, so the scheme of removing the grease with NaOH is not feasible.
Under the condition that a NaOH scheme is not feasible, daily-life grease removal detergents such as liquid detergent and liquid soap are supposed to be added, but the detergents may contain micro plastics originally, and on the premise of not introducing impurities, surfactants similar to the detergents are selected, so that sodium dodecyl sulfate (chemical purity, guangzhou Guanghua science and technology Co., ltd.) is selected and pre-experiment is carried out by using peanut oil.
1.2.3 experiments 7 and 8 are preliminary experiments of peanut oil, and in the experiment 7, a plurality of drops of water of peanut oil are directly added for suction filtration without adding lauryl sodium sulfate. The discovery shows that a large amount of peanut oil on the microporous filter membrane can not permeate the microporous filter membrane and block the filter device, and the suction filtration can not be continued even if ultrapure water is added; experiment 8 is that after the filtration, the sodium dodecyl sulfate solution is added, the peanut oil on the microporous filter membrane obviously permeates the filter membrane, a large amount of foam appears on the receiver, the added ultrapure water can permeate the microporous filter membrane quickly, and the blockage phenomenon in experiment 7 does not appear, so that the sodium dodecyl sulfate solution can effectively enable the liquid oil to permeate the microporous filter membrane with the diameter of 0.22 mu m in the preliminary experiment.
According to feasibility of peanut oil preliminary experiment, experiment 10 is designed, after oyster digestion is completed, 80 ℃ ultrapure water is added for dilution by 10 times, hot suction filtration is carried out, 80 ℃ lauryl sodium sulfate solution is added at the same time, liquid grease on the microporous filter membrane permeates the filter membrane, and finally, the microporous filter membrane is washed by a large amount of 80 ℃ ultrapure water.
Preparing a reagent:
(1) Sodium lauryl sulfate solution: 5g of sodium lauryl sulfate was added to 50mL of ultrapure water.
(2) 1mol NaOH solution: 2g of NaOH was dissolved in 50mL of ultrapure water.
Experiment 7: a beaker is taken, a plurality of drops of peanut oil are dripped into 50mL of ultrapure water, and the filtration is carried out by a 0.22 mu m microporous membrane.
Experiment 8: taking a beaker, dripping a plurality of drops of peanut oil into 50mL of ultrapure water, carrying out suction filtration by using a 0.22 mu m microporous filter membrane, and then adding a sodium dodecyl sulfate solution for degreasing.
Experiment 9: taking the digestion solution of 1.2.2, carrying out suction filtration by using a 0.22 mu m microporous filter membrane, and adding 1mol of NaOH solution after suction filtration.
Experiment 10: taking the digestion solution of 1.2.2, carrying out suction filtration by using a 0.22 mu m microporous filter membrane, and adding a sodium dodecyl sulfate solution for degreasing after suction filtration.
1.2.4 microscopy
The microfiltration membrane which is subjected to suction filtration in experiment 4 of 1.2.2 is put into a beaker filled with 20mL of ultrapure water, ultrasonic treatment is carried out for 30 seconds, then 20mL of saturated NaCl solution is added, and standing is carried out for 12 hours until layering is achieved. After 12 hours, 20. Mu.L of the supernatant of the solution was dropped onto a clean glass slide and observed in the bright field with a microscope (Olympus IX73 inverted microscope, olympus Co., ltd.).
In order to make the micro-plastics better observed, a density method is needed to gather part of the micro-plastics, and according to the sinking and floating phenomenon of the plastics in the table 4, a saturated NaCl solution is selected for density screening, because the density screening is safer and does not react with the nitric acid possibly remained after digestion is completed.
Table 4: sinking and floating phenomenon table of common plastics in solutions with different densities
Digestion was performed according to the procedure of experiment 4 of 1.2.2 and layering was performed using a saturated NaCl solution, and the upper layer solution was aspirated and observed under a 4-fold microscope, and the results are shown in FIG. 1, in which graphs (a), (b) and (c) are graphs under different fields of view. It can be seen that some block-shaped objects are regularly transparent, and are numerous, some have relatively large volumes, and the plastic features are obvious, and can be preliminarily judged to be micro-plastic.
1.3 quantitative determination of Microplasts in oysters
1.3.1 micro-Plastic staining experiments
Through preliminary observation of the micro-plastics, in order to further and accurately prove the existence of the micro-plastics and count the micro-plastics, amino groups are modified on the surface of the micro-plastics by a silanization technology, the modified amino groups are combined with fluorescein, and the micro-plastics are observed and counted under a fluorescence microscope.
(1) Pre-experiment for plastic dyeing: cutting a small piece of plastic on a plastic bag, cutting the plastic into pieces, putting the plastic pieces into a beaker filled with 9.5mL of methanol, adding 0.5mL of (3-aminopropyl) trimethoxysilane by using a liquid transfer gun, standing for 12 hours, then discarding the liquid in the beaker, washing for 2-3 times by using the methanol, completely removing the (3-aminopropyl) trimethoxysilane (reagent grade, adamas reagent company, ltd.), then adding the methanol and trace fluorescein isothiocyanate (FITC, qiyun biotechnology company, guangzhou City) for immersing the surface of the plastic, dyeing for about 4 hours, finally washing by using a large amount of ultrapure water, and storing for detection. The plastic was placed on a clean glass slide with tweezers and examined microscopically. As shown in fig. 2, the morphology of the plastic in the bright field of 4 times of the microscope was clearly observed, and if observed in the dark field, the plastic could not be observed; as shown in FIG. 3, the morphology of FITC-modified plastic in a dark field of 4 times of a microscope shows that the edge of the plastic is clearly observed to have obvious green fluorescence, which indicates that the silanization pre-experiment of the plastic is feasible.
(2) Micro plastic dyeing pre-experiment: taking 5mL of supernatant of a sample after standing for 12 hours in 1.2.4, performing suction filtration by using a 0.22-micron organic microporous filter membrane (Shanghai Xingya purifying material factory), washing the supernatant with methanol for 2-3 times, adding the organic microporous filter membrane into a beaker filled with the methanol, immersing the organic microporous filter membrane in the methanol, sealing the mouth of the beaker with a preservative film, performing ultrasonic treatment for 30 seconds, removing the organic microporous filter membrane after the ultrasonic treatment, adding trace Fluorescein Isothiocyanate (FITC) into the beaker, standing and dyeing for 4 hours, performing suction filtration, washing with a large amount of ultrapure water, and preserving in the ultrapure water for detection. Sucking 20 mu L of solution to be detected by using a pipette gun, sucking for 3 times, and performing a parallel experiment;
FIG. 4 is a diagram showing the morphology of the microplastic staining clearly observed according to this preliminary microplastic staining experiment. However, the scheme is not beneficial to counting the micro-plastics, the image is only a partial lens, and the manual counting error is larger, so that the concentration of a digested sample is improved, the volume absorbed in microscopic examination is reduced, so that liquid drops are completely presented under a single lens, and the counting is matched with computer software, therefore, a 1.3.1 experimental scheme (3) is designed, after the oyster is digested by nitric acid, 10mL of ultrapure water is directly filtered in a suction mode, a saturated NaCL solution is not added for layering, before the staining, the oyster is oscillated and shaken uniformly, 5mL of sample liquid is absorbed for staining, and 0.5 muL of sample liquid is absorbed in microscopic observation, so that the sample liquid can be completely observed in a single lens of a microscope.
(3) Micro plastic staining experiment: another oyster (9.5 g) was digested according to 1.2.2 experiment 4, filtered, and the resulting membrane was sonicated in 10mL of ultrapure water and stored. Oscillating, shaking uniformly, sucking 5mL of liquid, performing suction filtration by using a 0.22 mu L organic microporous filter membrane, washing for 2-3 times by using methanol, adding the organic microporous filter membrane into a beaker filled with the methanol, immersing the organic microporous filter membrane in the methanol, sealing the opening of the beaker by using a preservative film, performing ultrasonic treatment for 30 seconds, discarding the organic microporous filter membrane after the ultrasonic treatment, adding a trace amount of Fluorescein Isothiocyanate (FITC) into the beaker, standing and dyeing for 4 hours, performing suction filtration, washing by using a large amount of ultrapure water, and storing in the ultrapure water for detection.
In FIG. 5, (a), (b), and (c) are results of experiments for staining microplastics, and they were counted by computer software, and after digestion and suction filtration of 9.5g of oyster, 5mL of the sample solution was aspirated into 10mL of ultrapure water solution for staining, and 0.5. Mu.L was aspirated for microscopic examination. The number of plots (a) was 27, the number of plots (b) was 33, the number of plots (c) was 35, the average was 32, and calculated from the dilution factor:
the number of the oyster can be calculated to be 67368 per gram of micro plastic
1.4 component detection of Microplastic in oysters
1.4.1 Fourier Infrared Spectroscopy
(1) And (3) concentrating a sample: taking an oyster (13.44 g), digesting and filtering according to the method of 1.2.2 experiment 4, ultrasonically treating a microfiltration membrane subjected to suction filtration into 10mL of ultrapure water, standing for 12 hours, sucking 1mL of upper layer liquid into a centrifuge tube, drying at 80 ℃ to about 0.2mL, and storing for detection.
(2) Grinding a proper amount of potassium bromide solid particles into powder, preparing a tablet to be detected by using a tabletting mold (Tianjin Bo Tianshengda science and technology development Co., ltd.), drying in an infrared rapid dryer for 3 minutes, and performing Fourier infrared spectrum on-machine test (TENSOR 27 infrared spectrometer, germany Brookfield Infrared Instrument Co., ltd.).
(3) Grinding a proper amount of potassium bromide solid particles into powder, adding the sample liquid concentrated in the step (1), grinding the powder and the sample liquid together for a certain time, drying the powder in an infrared quick dryer for 3 minutes, preparing tablets to be detected by using a tabletting mold, drying the tablets in the infrared quick dryer for 1 minute, and performing Fourier infrared spectrum on-machine test.
FIG. 6 shows the IR spectrum of a sample, which shows a distinct characteristic peak only at 1400cm-1, and the identification of plastic documents [ plastic component [ J ] by IR spectrum, 2008,36 (6): 72-77] according to Liu Rongzhong et al (FIG. 7), polypropylene (PP) and polyvinyl chloride (PVC) both have characteristic peaks at 1400cm-1, whereas common plastics such as Polyethylene (PE), polystyrene (PS), polytetrafluoroethylene (PTFE) do not have characteristic peaks at this point. The micro plastic is plastic fragments decomposed in the sea under the action of factors such as light, heat, organisms and the like, the physical and chemical properties of the micro plastic can be changed accordingly, and the functional groups of the micro plastic are different from those of the conventional plastic and only have partial characteristic peaks of the original plastic. The sample has only a distinct characteristic peak at 1400cm-1 in the fourier spectrum, i.e. it may be a micro-plastic decomposed from polypropylene (PP) or polyvinyl chloride (PVC), or both, and therefore, for the oyster sample (13.44 g), the micro-plastic component herein is presumed to be polypropylene (PP) or polyvinyl chloride (PVC).
In addition, polypropylene (PP) and polyvinyl chloride (PVC) are common plastics in daily life. Polypropylene (PP) can be used in engineering accessories of automobiles, electrical appliances, textiles and the like, daily necessities, medical and health equipment, building materials and the like also appear, and polypropylene (PP) plastic products such as fishing ropes, packing belts, binding ropes, woven bags and the like are lost or discarded in the ocean due to operation and are common behaviors; similarly, polyvinyl chloride (PVC) is also available in building materials, consumer goods for daily use, and packaging materials, among which plastic bags, mineral water bottles, beverage bottles, and other edible plastic containers are available, and waste plastic bottles are more common in the ocean, not only is the moral awareness of people inadequate, but also an indispensable factor is present to feed plastic bottles into the ocean. The above examples, all given the micro-plastic containing ingredient polypropylene (PP) or polyvinyl chloride (PVC) in the oysters herein, are a more complete rationality inference.
1.4.2 Electron microscopy
Taking the sample to be detected stored in the step (1) in the step 1.4.1, and sending the sample to be detected for detection. FIG. 8 shows an image of a sample (13.44 g) under an electron microscope (S4800 scanning electron microscope, hitachi, ltd.) (a) (c) shows that both images have a characteristic of a plastic fragment and a broken plastic fragment, and (b) shows a plastic fragment in the form of a film, which may be decomposed from a plastic bag, and further proves the fact that the sample contains Polychloroprene (PVC) in 4.3.1.
According to the research, oysters are used as experimental objects to perform a series of extraction experiments such as wet digestion and degreasing and optimization of the extraction experiments, extracted micro-plastics are dyed by a silanization technology, the number of the micro-plastics is preliminarily estimated, and the type and appearance characteristics of the micro-plastics are found by a Fourier infrared spectrum technology and an electron microscope. The results of the study are as follows:
(1) The optimized extraction scheme is that about 12g of oyster sample is soaked in 25mL of HNO3 for 12 hours, heated for 2 hours, washed with 80 ℃ sodium dodecyl sulfate solution added with surfactant while hot, washed cleanly with 80 ℃ ultra-pure water, and if concentration and calculation are needed, ultrasonic treatment is carried out on the oyster sample in 10mL of ultra-pure water, and if the oyster sample is separated, 10mL of saturated sodium chloride solution can be added.
(2) The best staining protocol, cancellation of the finished samples, in 10mL of ultrapure water ultrasound, and suction filtration to organic microporous filter membrane, according to the 19 ratio of methanol and (3-aminopropyl) trimethoxysilane modification of amino to the micro plastic, again suction filtration, with trace Fluorescein Isothiocyanate (FITC) staining, and fluorescence microscope observation. The result of this experimental oyster is 67368/g micro plastic.
(3) The best characterization scheme is that a sample after the completion of the chemical modification is cancelled, ultrasonic treatment is carried out in 10mL of ultrapure water, 1-2 mL of the sample is dried at 80 ℃, the sample is concentrated, and Fourier infrared spectrum detection and electron microscope characterization are carried out. The experimental result shows that the oysters detected by the experiment contain polypropylene (PP) or polyvinyl chloride (PVC) or both.
The above description is only a preferred embodiment of the present invention and should not be taken as limiting the invention. Any modification, replacement or improvement that comes within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A method of detecting a micro-plastic in a sample of a biological organism, comprising:
s100, a step of extracting the micro-plastic from the biological sample, which comprises:
s110, digestion: soaking the biological sample in 50-70% nitric acid at room temperature for 6-18 hr, heating to 60-95 deg.C and maintaining for 1-4 hr to obtain digested biological sample;
s120, degreasing: washing and filtering the digested organism sample obtained in the step S110 with a surfactant solution at the temperature of 60-95 ℃ while the digested organism sample is hot to obtain degreased micro-plastic;
s130, washing and filtering the degreased micro plastic obtained in the step S120 by using water at the temperature of 60-95 ℃ to obtain a cleaned micro plastic;
s200, dyeing the cleaned micro plastic obtained in the step S100, wherein the dyeing comprises the following steps: contacting the micro-plastic with aminosiloxane to obtain the micro-plastic with amino, and then contacting the micro-plastic with fluorescein isothiocyanate to obtain dyed micro-plastic, wherein the aminosiloxane is (3-aminopropyl) trimethoxysilane, and methanol and (3-aminopropyl) trimethoxysilane are added into the micro-plastic according to the proportion of 19;
s300, the dyed microplastic obtained in step S200 is observed with a microscope, and the microplastic particles are counted.
2. The method for detecting micro plastic in a biological sample according to claim 1, wherein the step S100 further comprises: and adding the obtained cleaned micro plastic into water, and then carrying out ultrasonic treatment to obtain a micro plastic suspension.
3. The method for detecting a microplastic in a biological sample according to claim 1, wherein the suction filtration is a suction filtration with a microfiltration membrane.
4. The method for detecting micro plastic in a biological sample according to claim 1, wherein the step S100 further comprises:
s140, grading: grading the micro plastic by using solutions with different densities according to the densities; the solution is selected from the group consisting of a density of 1g/cm 3 Water of 1.19g/cm density 3 Saturated sodium chloride aqueous solution of (2) having a density of 0.91g/cm 3 58.4% ethanol aqueous solution with a density of 0.925g/cm 3 55.4% ethanol aqueous solution, and a density of 1.27g/cm 3 27.5% calcium chloride in water.
5. The method for detecting a microplastic in a biological sample according to any one of claims 1 to 4, wherein the surfactant is sodium lauryl sulfate.
6. The method for detecting a microplastic in a sample of an organism according to any one of claims 1 to 4, wherein the organism is an oyster.
7. The method for detecting a microplastic in a biological sample according to any one of claims 1 to 4, further comprising:
and S400, carrying out infrared spectrum detection or electron microscope characterization on the cleaned micro plastic obtained in the step S100.
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