Sterile micro-plastic particle and preparation method thereof

Abstract

The invention provides sterile micro-plastic particles and a preparation method thereof, and relates to the technical field of ecological toxicology, and the preparation method comprises the following steps: disinfecting a workbench with alcohol, and keeping sterile conditions for operation; weighing the micro-plastic particles, weighing a proper amount of the micro-plastic particles, putting the micro-plastic particles into a sterile centrifuge tube, spraying alcohol to the outside of the centrifuge tube for disinfection, putting the centrifuge tube into a super clean bench, and placing the centrifuge tube in a shading manner for 3-5 days; adding the micro-plastic particles in the centrifugal tube into an F12 culture medium containing a double antibody which is an antibiotic solution formed by mixing penicillin and streptomycin according to a certain proportion, blowing and beating the culture medium, centrifuging, and discarding supernate; adding into an F12 culture medium containing double antibodies, and repeatedly cleaning for 3-5 times; and preparing the required concentration by using a complete culture medium.

Description

Technical Field

[0001] The present invention relates to the field of ecotoxicology technology, and in particular to a method for preparing sterile microplastic particles. Background Art

[0002] Ecotoxicology is a science that studies the effects of toxic substances on ecosystems and organisms and their interactions. It focuses on the behavior, bioaccumulation, ecological effects and risk assessment of chemical pollutants in the environment, aiming to reveal the potential harm of toxic substances to biodiversity and environmental quality, and provide a scientific basis for environmental management and pollution prevention. Toxicology research models are divided into in vivo models and in vitro models. In vitro models mainly study the damage of drugs to cells to explore drug toxicity and mechanisms.

[0003] Microplastics are a new type of pollutant that can reach almost all parts of the world through atmospheric deposition. Microplastics can enter the body in a variety of ways, such as inhalation or ingestion. Currently, many scholars are studying the toxicology and mechanisms of microplastics to explore the damage of this pollutant to the ecological environment, wildlife and even human health. The cellular toxicology of microplastics has become a hot topic in the scientific community.

[0004] However, there are still objections to the way microplastics are exposed to cells. Microplastic powder raw materials need to go through steps such as weighing and configuration. Most experiments use high-pressure sterilization, ultraviolet disinfection and other methods to achieve sterile conditions for cell experiments. However, current studies have shown that these two methods will change the physical and chemical properties of microplastics before they are exposed to cells, causing microplastics to age, such as cracking or deformation, or be used to study special functional group changes. Some specially treated microplastics will cause particles to stick together after high pressure, making it impossible to correctly detect the true impact of microplastics on cells. Summary of the invention

[0005] The purpose of the present invention is to provide a sterile microplastic particle and a preparation method thereof, so as to solve the problem that the traditional sterilization method causes inaccurate experimental results when the microplastic configuration is exposed to a cell solution.

[0006] To solve the above problems, the present invention first provides a method for preparing sterile microplastic particles, comprising: disinfecting the workbench with alcohol and maintaining sterile conditions; weighing the microplastic particles, weighing an appropriate amount and placing them in a sterile centrifuge tube, spraying the outside of the centrifuge tube with alcohol for disinfection, placing it in an ultra-clean bench, and shielding the centrifuge tube from light for 3-5 days; adding the microplastic particles in the centrifuge tube to an F12 culture medium containing double antibodies, wherein the double antibodies are an antibiotic solution of penicillin and streptomycin mixed in a certain proportion, blowing the culture medium, and discarding the supernatant after centrifugation; then adding them to the F12 culture medium containing double antibodies, and repeating washing 3-5 times; and configuring them to the required concentration with a complete culture medium.

[0007] With such an arrangement, the preparation method avoids the effects of traditional ultraviolet sterilization or high-pressure sterilization on aging of microplastic particles. The centrifuge tube containing the microplastics is disinfected with alcohol and placed on a clean bench for several days. Bacteria are not easy to survive in a dry environment without nutrient solution. After the sterilization is completed, the cells are cultured and washed with cell culture fluid, and then the microplastic particles in the required proportion are configured. Such treatment will not affect the chemical properties of the microplastics, produce harmful substances that affect the experimental results, and solves the defects of the prior art.

[0008] Furthermore, when the centrifuge tube is placed on the clean bench, its outside is wrapped with tin foil.

[0009] Furthermore, 1 ml of F12 culture medium contained 1% double antibody.

[0010] Furthermore, the complete culture medium is prepared by mixing F12 culture medium, double antibody and serum in a certain proportion, and each 1 ml of F12 culture medium contains 10% serum and 1% double antibody.

[0011] The present invention also provides sterile microplastic particles, which are made by the preparation method of sterile microplastic particles of the above technical solution.

[0012] Furthermore, toxicological experiments were used to study the cytotoxicity and mechanisms of microplastics. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0014] Figure 1 This is a picture of AML12 cells and plastic particles that were cultured for 72 hours after being treated by the preparation method provided in Example 1 of the present invention;

[0015] Figure 2 This is the SEM image of polystyrene microplastics before autoclaving;

[0016] Figure 3 This is the SEM image of polystyrene microplastics after autoclaving;

[0017] Figure 4 This is the SEM image of amino-modified polystyrene microplastics before autoclaving;

[0018] Figure 5 This is the SEM image of amino-modified polystyrene microplastics after autoclave sterilization. DETAILED DESCRIPTION

[0019] In order to make the above-mentioned objects, features and advantages of the present invention more clearly understood, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0020] Example 1 Preparation method of sterile microplastic particles

[0021] At present, when conducting toxicological experiments in this field, microplastic particles are usually sterilized by ultraviolet light or high pressure sterilization, which will cause the microplastics to age or deform, and easily produce harmful substances, adsorb harmful substances, change the chemical properties of special functional groups, and some will adhere, thus affecting the experimental operation and causing errors in the experimental results. The results are inaccurate and cannot reflect real problems. For example, Figure 2 and 3 Comparison of SEM images of polystyrene microplastics (PS-MPs) particles before and after autoclaving. Figure 4 and 5 The SEM image comparison of amino-modified polystyrene microplastics (PS-NH2-MPs) particles before and after high-pressure sterilization clearly shows the impact of current traditional sterilization methods on microplastic particles.

[0022] This embodiment provides a method for preparing sterile microplastic particles to solve the problem of inaccurate experimental results caused by traditional sterilization methods when microplastic configurations are exposed to cell solutions.

[0023] The specific steps of the preparation method are as follows:

[0024] First, wipe and disinfect the workbench with alcohol to maintain sterile conditions;

[0025] Open the prepared microplastic particles for weighing. You can use an electronic balance to weigh the purchased finished microplastics and the collected microplastic powder or particles. Put the appropriate amount into a sterile 1.5ml centrifuge tube. Spray the outside of the centrifuge tube with alcohol for disinfection and put it into the clean bench.

[0026] When placed, the centrifuge tube is shielded from light, preferably wrapped with tin foil, and placed in a dark place for 3-5 days;

[0027] Add the microplastic particles in the centrifuge tube to the F12 medium containing double antibodies. The double antibodies are antibiotic solutions of penicillin and streptomycin mixed in a certain proportion. 1 ml of F12 medium contains 1% double antibodies. Pipette the medium and discard the supernatant after centrifugation.

[0028] Then add F12 medium containing double antibodies and repeat washing 3-5 times;

[0029] The required concentration is prepared with complete culture medium, which is a mixture of F12 culture medium, double antibody and serum in proportion. Each 1 ml of F12 culture medium contains 10% serum and 1% double antibody. In this embodiment, fetal bovine serum is used.

[0030] This preparation method avoids the effects of traditional ultraviolet sterilization or high-pressure sterilization on aging of microplastic particles. After the microplastic particles are disinfected with alcohol, they are placed on a clean bench for several days. Bacteria are not easy to survive in a dry environment without nutrient solution. After the sterilization treatment is completed, the particles are cultured and washed with cell culture fluid, and then the microplastic particles in the required proportion are configured. Such treatment will not affect the chemical properties of the microplastics, produce harmful substances that affect the experimental results, and solve the defects of the prior art.

[0031] Example 2 Sterile microplastic particles

[0032] This embodiment provides a sterile microplastic particle, which is mainly used for toxicological experiments to study the cytotoxicity and mechanism of microplastics, and is made by the preparation method of the sterile microplastic particles in Example 1.

[0033] This embodiment sets up an experiment on the effect of different sterilization methods on plastic particles. A total of three groups are set up for treatment. Three six-well plates are used for operation. The first group only has cells, the second group adds cells and treated microplastics, and the third group adds cells and untreated microplastics. The concentration of microplastics in the second and third groups is the same; the cell density is the same in the above three groups of experiments. For example, 20W AML12 cells are added. In order to avoid damage to cells by microplastics, a small amount of large-particle microplastics needs to be added. 2 ml of culture medium is used, and observation is made after 72 hours of culture.

[0034] The observation results are as follows: the culture medium of the first and second groups is clear, not turbid, and orange-yellow, while the culture medium of the third group is turbid and yellow; microscopic observation of the growth of the cells of the first and second groups is the same, and the cell morphology is the same, proving that there is no contamination. The culture results of the second group are as follows: Figure 1 As shown, the third group was obviously contaminated, which proves that the culture results of the sterile microplastic particles prepared by this method are good and can be used for toxicological experimental research without affecting the accuracy of the results.

[0035] Finally, it should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the presence of other identical elements in the process, method, article or device including the elements.

[0036] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0037] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing sterile microplastic particles, characterized in that: include: Disinfect the workbench with alcohol and maintain sterile conditions; Weigh the microplastic particles, take an appropriate amount and put them into a sterile centrifuge tube, spray the outside of the centrifuge tube with alcohol for disinfection, put it into a clean bench, and place the centrifuge tube in a dark place for 3-5 days; Add the microplastic particles in the centrifuge tube to the F12 culture medium containing double antibodies, which are antibiotic solutions of penicillin and streptomycin mixed in a certain proportion, blow the culture medium, and discard the supernatant after centrifugation; Then add F12 medium containing double antibodies and repeat washing 3-5 times; Use complete medium to prepare the desired concentration.

2. The method for preparing sterile microplastic particles according to claim 1, characterized in that: When placing the centrifuge tube on the clean bench, wrap its outside with tin foil.

3. The method for preparing sterile microplastic particles according to claim 1, characterized in that: 1 ml of F12 culture medium contains 1% double antibody.

4. The method for preparing sterile microplastic particles according to claim 1, characterized in that: The complete culture medium is prepared by mixing F12 culture medium, double antibody and serum in a certain proportion, and each 1 ml of F12 culture medium contains 10% serum and 1% double antibody.

5. A sterile microplastic particle, characterized in that: Made by the preparation method of the sterile microplastic particles described in any one of claims 1-4.

6. The sterile microplastic particles according to claim 5, characterized in that Used in toxicology experiments to study the cytotoxicity and mechanisms of microplastics.

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