Method for rapidly detecting nano plastic in food and environmental sample based on self-made nano magnetic beads

Abstract

The invention discloses a method for rapidly detecting nano plastic in food and environmental samples based on self-made nano magnetic beads, and belongs to the technical field of pollution detection. The method comprises the following steps: firstly, preparing nano magnetic beads, reacting FeCl2 and FeCl3 in an ammonia water environment, washing, activating, and freeze-drying to obtain the nano magnetic beads; preparing a fluorescent nano-plastic standard curve, dyeing by using Nile red and detecting and drawing by using a microplate reader; and finally, adsorbing the nano plastic in the sample by using nano magnetic beads, performing fluorescence labeling after desorption, detecting by using a microplate reader, and substituting into a standard curve to obtain the concentration. The nano magnetic beads are uniform in particle size and good in magnetic performance, the adsorption rate of the nano plastic is larger than or equal to 80%, fluorescence labeling and microplate reader detection are combined, large instruments are not needed, operation is easy and convenient, and the nano magnetic beads are suitable for rapid detection of the nano plastic in food and environmental samples.

Description

Technical Field

[0001] This invention belongs to the field of pollution detection and analysis technology, specifically relating to a rapid detection method for nanoplastics in food and environmental samples based on self-made nanomagnetic beads. Background Technology

[0002] Plastic debris is a major pollutant in aquatic ecosystems today, attracting worldwide attention due to its resistance to degradation and its potential. Due to the massive consumption of plastics and poor management of plastic waste, an estimated 8 million tons of plastic leak from land sources into rivers and oceans globally each year. Large pieces of plastic inevitably degrade into large quantities of macroplastics (5-20 mm), microplastics (<5 mm), and nanoplastics (1-1000 nm) through physical decomposition and weathering (e.g., chemically induced degradation).

[0003] Due to their extremely small size, nanoplastics possess unique physical and chemical properties, such as a large specific surface area and high surface activity. This makes their behavior in the environment and their impact on organisms different from that of conventionally sized plastics. They may more easily enter organisms and potentially trigger various ecotoxicological effects, posing a threat to the environment and biological health. Nanoplastics in the environment can enter the human body through various exposure routes, such as through the respiratory system, causing adverse effects. In addition, some edible plants, such as fruits and vegetables, may accumulate nanoplastics from soil and irrigation water during their growth, and some processed meat products, such as ham, can also be contaminated with nanoplastics during processing. Therefore, detecting plastic debris in the environment is of great significance for protecting ecosystems and human health.

[0004] Currently, the main method used to remove nanoplastics is traditional filtration. However, due to the physical limitations of filter pores, it is not suitable for removing nanoplastics. Rapid adsorption materials are an emerging and promising method for pollutant removal. However, their low adsorption capacity and complex processes make them unsuitable for removing microplastics from the environment.

[0005] Traditional methods for detecting nanoplastics mainly involve scanning electron microscopy (SEM), but these methods are expensive due to the large size of the equipment, the need for a stable power supply and specific environmental conditions. There is an urgent need for a convenient, fast, and low-cost method for rapid detection of nanoplastics. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a rapid detection method for nanoplastics in food and environmental samples based on self-made magnetic nanobeads. This method achieves efficient adsorption of nanoplastics by preparing high-performance magnetic nanobeads. This method combines separation with fluorescent labeling and ELISA reader detection to achieve rapid quantitative analysis. It is simple to operate, low in cost, and highly efficient, making it suitable for batch screening of food and environmental samples.

[0007] The technical solution of the present invention is as follows: A rapid detection method for nanoplastics in food and environmental samples based on self-made magnetic nanobeads includes the following steps: S1. Preparation of nanomagnetic beads: (1) Add 1.5g FeCl2, 4.05g FeCl3 and 75mL ultrapure water to a three-necked flask, and react with a magnetic stirrer at 70-80℃ in a 37.5mL ammonia environment for 50-80min. Nitrogen gas is introduced for protection at a flow rate of 0.5g / min. After the solution turns deep orange, add 0.5mL oleic acid and continue to react at 60-70℃ for 40-50min. (2) Perform magnetic separation on the reaction mixture. First wash with 100 mL of deionized water 2-3 times, then wash with 80 mL of anhydrous ethanol once, and then perform ultrasonic treatment for 5-10 min before magnetic separation again. (3) Add 30 mL of 0.1 g / mL 2-(n-morpholine) ethanesulfonic acid-hydrate solution sequentially, and sonicate at 30 °C for 20 min with an ultrasonic power of 0.5 W / cm². 2 After magnetic separation, 40 mL of 0.1 g / mL 2-(n-morpholine)ethanesulfonic acid-hydrate solution was added, and the mixture was sonicated at 30 °C for 30 min. Then, 70 mL of 0.04 g / mL carbodiimide solution was added, and the mixture was incubated on a shaker at 25 °C for 40 min. After freeze-drying, nano-magnetic bead powder was obtained. S2. Preparation of standard curves for fluorescent nanoplastics: (1) Dissolve 1g of Nile Red dye in 10mL of chloroform under light-protected conditions to prepare Nile Red dye solution and store it in the dark. (2) Weigh different masses of standard nanoplastics and disperse them in a 9 mL mixture of ultrapure water and dimethyl sulfoxide to prepare nanoplastic solutions with concentrations of 0, 0.1, 0.2, 0.4 and 0.8 μg / mL; (3) At 75°C, add 1 mL of Nile Red dye solution to each concentration of nanoplastic solution, heat for 30 min and then immediately cool with ice water to obtain fluorescent nanoplastics; (4) Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance of fluorescent nanoplastics. The excitation wavelength is 543 nm and the emission wavelength is 620 nm. Plot a standard curve based on the relationship between absorbance and concentration. S3. Detection of nanoplastics in samples: (1) Add the nanomagnetic beads obtained in step S1 to food or environmental samples and incubate them at 37°C and pH 3-12 for 30-180s. The adsorption of nanoplastics by the nanomagnetic beads is achieved by applying an external magnetic field. (2) Adjust the pH value of the nanomagnetic beads and nanoplastic composite to desorb the nanoplastic. When pH>10, the desorption effect is more obvious. The pH value can be adjusted by slowly adding 25% concentrated ammonia or 1mol / L sodium carbonate to desorb the nanoplastic. Collect the desorbed nanoplastic and perform fluorescent labeling according to the method in (2) and (3) of step S2. (3) Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance value of the labeled sample, and substitute it into the standard curve to calculate the concentration of nanoplastics in the sample.

[0008] Further, the concentrations and volumes of 2-(n-morpholine)ethanesulfonic acid hydrate and carbodiimide in step S1(3) are as follows: the concentration of 2-(n-morpholine)ethanesulfonic acid hydrate is 0.1 g / mL and the volume is 70 mL, and the concentration of carbodiimide is 0.04 g / mL and the volume is 70 mL.

[0009] Furthermore, in step S2 (3), the heating method is a constant temperature water bath, and the enzyme-linked immunosorbent assay (ELISA) detection must be completed within 30 minutes after cooling.

[0010] Furthermore, in step S3(1), the mass-to-volume ratio of the nanomagnetic beads to the sample is 1:2.5; each gram of nanomagnetic beads can adsorb 2.5 grams of nanoplastics.

[0011] Furthermore, in step S3, the required concentration of Nile Red dye for the preparation of the fluorescent nanoplastics is 10 μg / mL.

[0012] Furthermore, the ratio of ultrapure water to dimethyl sulfoxide in step S2 (2) is 1:1.

[0013] Furthermore, the food samples include liquid food, solid food, semi-solid food, and pre-prepared dishes; the environmental samples include soil and water.

[0014] Furthermore, the nanomagnetic beads obtained in step S1 have a particle size ≤30nm, are regularly spherical, and have amino groups on their surface.

[0015] Furthermore, in step S3, the adsorption rate of the nanomagnetic beads on the nanoplastics is ≥80%.

[0016] Compared with the prior art, the beneficial effects of the present invention are: 1. The nanomagnetic beads prepared by this invention have a particle size ≤30nm, are regularly spherical, have amino groups on their surface, and have excellent magnetic properties. They can be quickly separated by an external magnetic field and have an adsorption rate of ≥80% for nanoplastics in food and environmental samples, thus solving the problem of low efficiency of traditional separation methods.

[0017] 2. In the manufacturing process of the nanomagnetic beads of the present invention, organic matter is added to give them functional groups, which can be combined with nanoplastics through interaction forces to achieve efficient and rapid adsorption of nanoplastics.

[0018] 3. The composite of nanomagnetic beads and nanoplastics of the present invention can rapidly aggregate around a magnet under an external magnetic field, thereby removing nanoplastics from environmental and food samples.

[0019] 4. This invention uses Nile Red dye to impart fluorescence to nanoplastics, enabling quantitative detection of nanoplastic particles.

[0020] 5. This invention combines fluorescent labeling technology with enzyme-linked immunosorbent assay (ELISA) detection, eliminating the need for large instruments, simplifying operation, and shortening the detection cycle, making it suitable for rapid analysis of batch samples.

[0021] 6. The detection range of this invention covers food samples such as liquid food, solid food, semi-solid food, and pre-cooked dishes, as well as environmental samples such as soil and water, and is applicable to a wide range of scenarios.

[0022] 7. This invention uses magnetic nanobeads to adsorb nanoplastics in samples, then performs fluorescent labeling, and finally substitutes the results into a standard curve, which enables rapid detection of nanoplastics. Attached Figure Description

[0023] Figure 1 shows the morphology and structural characterization of the nanomagnetic beads of the present invention; wherein: A is a scanning electron microscope image of the nanomagnetic beads of the present invention; B is a Fourier transform infrared spectrum of the nanomagnetic beads; Figure 2 shows the adsorption of nanoplastics by the nanomagnetic beads of the present invention; wherein: A is a scanning electron microscope image of nanoplastics adsorbed by the nanomagnetic beads; B is the adsorption rate of nanoplastics by the nanomagnetic beads. Figure 3 shows the optimized conditions for the adsorption of nanoplastics by the nanomagnetic beads of the present invention; where: A represents the study of adsorption time; B represents the study of pH value; Figure 4 This demonstrates the adsorption of nanoplastics in the environment by the nanomagnetic beads of this invention; Figure 5 This invention demonstrates the adsorption of nanoplastics in liquid food by the nanomagnetic beads of this invention; Figure 6 This invention demonstrates the adsorption of nanoplastics in different types of solid food by the nanomagnetic beads of this invention; Figure 7 This demonstrates the adsorption of nanoplastics in two types of pre-cooked dishes under different heating methods by the nanomagnetic beads of this invention; Figure 8 This invention demonstrates the adsorption of nanoplastics in three food packaging materials by the nanomagnetic beads of the present invention under different temperature treatments; Figure 9This invention describes the fabrication of fluorescent nanoplastics. Figure 10 This describes the preparation of the standard curve for fluorescent nanoplastics according to the present invention. Detailed Implementation

[0024] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0025] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0026] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0027] The technical solution of the present invention will be described in detail below with reference to the embodiments, but the scope of protection is not limited thereto.

[0028] Example 1: Preparation and characterization of nanomagnetic beads Experimental methods FeCl2, FeCl3, and ultrapure water were added to a three-necked flask and reacted in an ammonia environment on a magnetic stirrer. After the solution turned deep orange, oleic acid was added dropwise to continue the reaction. Nitrogen gas was purged at a flow rate of 0.5 g / min during the reaction. The resulting mixture was magnetically separated, washed with deionized water and anhydrous ethanol, and then sonicated. 2-(n-morpholine)ethanesulfonic acid hydrate and carbodiimide were added separately and mixed thoroughly. The mixture was then activated on a mixer and finally freeze-dried to obtain nano-magnetic beads powder. The morphology of the nano-magnetic beads was characterized using scanning electron microscopy, and the structure was characterized using Fourier transform infrared spectroscopy.

[0029] Experimental results The prepared magnetic nanobeads have a particle size of less than 30 nm, uniform particle size, and a regular spherical shape (see...). Figure 1A Structurally, amino groups are attached to the magnetic beads to prepare amino-based magnetic nanobeads (see...). Figure 1B ).

[0030] Example 2: Adsorption of nanoplastics by nanomagnetic beads Experimental methods A standard nanoplastic solution of 40 μg / mL was prepared. 10 mL of the nanoplastic solution was placed in a beaker, and 2 mg of magnetic nanobeads were added. The mixture was incubated at 37°C for 1 min. After adsorption, magnetic separation was performed using an external magnetic field, and the supernatant and precipitate were collected separately. The nanobead-nanoplastic composite in the precipitate was resuspended, and the pH of the resuspended solution was adjusted to separate the nanobeads and nanoplastics. The adsorption was then observed under a microscope and a scanning electron microscope.

[0031] (1) Count the unadsorbed large microplastic particles (>1μm) under a microscope and calculate the adsorption rate of the magnetic nanoparticles. The specific method is as follows: Use a hemocytometer with a 25×16 counting chamber. Before counting, perform a microscopic inspection to ensure that there are no contaminants in the counting chamber that may affect the experimental results. Take 100μL of the collected supernatant to the edge of the coverslip, allowing the solution to permeate into the counting chamber on its own. Let it stand for a moment and count the microplastics under a stereomicroscope at 40x magnification. For microplastics located on the grid lines, follow the principle of "counting the top but not the bottom, counting the left but not the right". The number of microplastics is calculated according to Formula 1: Number of nanoplastics per mL = Average number of nanoplastics per cell × 400 × 10⁴ × Dilution factor (Formula 1) The adsorption rate of nanomagnetic beads on nanoplastics is calculated according to Formula 2: Adsorption rate (%) = ×100% Formula 2 (2) The number of small nanoplastic particles (<1 μm) was further analyzed by scanning electron microscopy. To count the nanoplastics in the supernatant, a nanoplastic suspension was dropped onto a glass slide. The number of unbound nanoplastics in five regions was quantified by the following method, and the results are expressed as the average number of nanoplastics not bound to the magnetic beads. The adsorption rate of the magnetic beads was calculated using Formula 2.

[0032] Experimental results The results are shown in Figure 1. Scanning electron microscopy revealed that the magnetic nanobeads were adsorbed onto the surface of the nanoplastic (see Figure 1). Figure 2A Microscopic counting results showed that the adsorption rate of the magnetic nanobeads on the nanoplastics reached over 80% within 1 minute (see...). Figure 2B ).

[0033] The above results show that the magnetic nanobeads prepared by this invention can be adsorbed on the surface of nanoplastics in a very short time with an extremely high adsorption rate, thus enabling rapid and efficient removal of nanoplastics.

[0034] Example 3: Optimization of conditions for the adsorption of nanoplastics by magnetic nanobeads 1. Experimental Methods (1) Adsorption time The standard microplastic concentration was set at 40 μg / mL and pH=7. 2 mg of magnetic nanobeads were added to the nanoplastic solution and adsorption was carried out at 37°C for 30, 60, 90, 120, 150 and 180 s. After adsorption, magnetic separation was performed, the supernatant was collected, and the nanoplastics were counted using the method in Example 2.

[0035] (2) Determination of adsorption pH value The pH values ​​of the nanoplastic solutions were adjusted to 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. 2 mg of nanomagnetic beads were added to the nanoplastic solutions with different pH values ​​and adsorbed at 37°C for 1 min. After adsorption, magnetic separation was performed, and the supernatant was collected. The nanoplastics were counted using the method described in Example 2.

[0036] Experimental results (1) Effect of adsorption time on adsorption rate The results are shown in Figure 3. The adsorption rate of the magnetic nanobeads on the nanoplastics reached over 90% within 30 seconds. The adsorption rate increased with time, then plateaued at 150 and 180 seconds, at which point it reached over 95% (see Figure 3). Figure 3A Overall, the nanomagnetic beads achieved near-complete adsorption of nanoplastics in a very short time, indicating that the entire adsorption process is extremely efficient.

[0037] (2) Effect of pH value on adsorption rate Depend on Figure 3B It can be seen that when pH>7, the adsorption rate of nanomagnetic beads on nanoplastics is relatively high. When the pH value is 9, 10, and 11, the adsorption rate of nanomagnetic beads reaches 100%, which indicates that the adsorption effect of nanomagnetic beads on nanoplastics is better in a neutral to slightly alkaline environment.

[0038] In summary, the optimal adsorption time for the nanomagnetic beads is 150 seconds, and the optimal adsorption pH is 9.

[0039] Example 4: Removal of nanoplastics from the environment using nanomagnetic beads (taking a soil sample as an example) 1. Experimental Methods (1) Extraction of nanoplastics from soil Weigh 50g of dried soil sample into a 500mL Erlenmeyer flask, add 300mL of saturated NaCl solution, vortex on a vortex apparatus for 10min, let stand for 24h, collect the supernatant, repeat the above operation 2-3 times, mix the supernatants collected three times, add 10mol / L NaOH solution and digest on a shaker at 60℃ for 24h.

[0040] (2) Adsorption of nanoplastics in soil by nanomagnetic beads 5 mg of magnetic nanobeads were added to the supernatant and incubated on a shaker at 37 °C for 180 s. After adsorption, magnetic separation was performed, and the supernatant was collected. The supernatant was then filtered through organic filter membranes with pore sizes of 0.22 μm, 0.05 μm, and 0.03 μm, and the filter membranes were placed in clean glass petri dishes. The membranes were then dried in an oven at 50 °C, sputter-coated with gold, and observed. The filter membranes were then observed under a microscope, and the adsorption rate of the magnetic nanobeads on the nanoplastics was calculated.

[0041] 2. Experimental Results like Figure 4 As shown, the adsorption rate of nanoplastics in soil by the nanomagnetic beads reached over 80% within 3 minutes, indicating that the nanomagnetic beads have a good adsorption effect on nanoplastics in the environment.

[0042] Example 5: Adsorption of nanoplastics in liquid food by nanomagnetic beads Experimental methods In this embodiment, bottled beverages were selected as the research object. 100 mL of beverage sample was measured into a beaker, and 2 mg of nanomagnetic beads were added to the beverage sample. The mixture was incubated on a shaker at 37 °C for 180 s. After adsorption, magnetic separation was performed, and the supernatant was collected and filtered through a 0.22 μm microporous membrane. The filter membrane was observed under a microscope, and five regions were selected to count the nanoplastics. The adsorption rate of the nanomagnetic beads on the nanoplastics was calculated according to Formula 1.

[0043] Experimental results The results are as follows Figure 5 As shown, the microscopic counting results indicate that the adsorption rate of nanoplastics in liquid food by the magnetic nanobeads reaches more than 80% within 180s, which shows that the magnetic nanobeads have a good adsorption effect on nanoplastics in liquid food.

[0044] Example 6: Adsorption of nanoplastics in solid food by nanomagnetic beads Experimental methods (1) Extraction of nanoplastics from solid foods In this embodiment, four common foods were selected: apples, potatoes, steamed buns, and ham sausages. After freeze-drying, 5g of each sample was weighed into a pre-cleaned 500mL Erlenmeyer flask, and 100mL of digestion reagent was added. The digestion reagent was prepared by mixing 65%-68% HNO3 and 30% H2O2 in a 4:1 ratio. The samples were digested in a 50℃ water bath for 6 hours, with the Erlenmeyer flask shaken for 1 minute every 30 minutes. When the solution became clear and there was no obvious organic residue, the food was considered to be completely digested.

[0045] (2) Adsorption of microplastics in sediments by nanomagnetic beads The digestion solution was diluted to 500 mL with deionized water, 10 mg of magnetic nanobeads were added, and the mixture was incubated on a shaker at 37 °C for 180 s. After adsorption, magnetic separation was performed, the supernatant was collected, and the solution was filtered through a 0.22 μm microporous membrane. The membrane was observed under a microscope, and five regions were selected to count the microplastics. The adsorption rate of the magnetic nanobeads on the nanoplastics was calculated according to Formula 1.

[0046] 2. Experimental Results The results are as follows Figure 6 As shown, the microscopic counting results indicate that the adsorption rate of nanoplastics in different solid foods by the magnetic nanobeads reached over 80% within 180s, which shows that the magnetic nanobeads have a good adsorption effect on nanoplastics in different types of solid foods.

[0047] Example 7: Adsorption of nanoplastics in semi-solid food by nanomagnetic beads 1. Experimental Methods This embodiment uses jam as the research object. 50g of jam sample was weighed into a 500mL Erlenmeyer flask, 300mL of saturated NaCl solution was added, and the mixture was vortexed for 10min. After standing for 24h, the supernatant was collected. This process was repeated 2-3 times. The supernatants collected three times were mixed, and 10mol / L NaOH solution was added. The mixture was digested on a shaker at 60℃ for 24h. 5mg of magnetic nanobeads were added to the supernatant, and the mixture was incubated on a shaker at 37℃ for 180s. After adsorption, magnetic separation was performed, and the supernatant was collected. The supernatant was then filtered through a 0.22μm microporous membrane. The membrane was observed under a microscope, and five regions were selected to count the nanoplastics. The adsorption rate of the magnetic nanobeads on the nanoplastics was calculated using Formula 1.

[0048] 2. Experimental Results like Figure 7 As shown, the adsorption rate of nanoplastics in jam by the nanomagnetic beads reached over 80% within 3 minutes, indicating that the nanomagnetic beads have a good adsorption effect on nanoplastics in semi-solid foods.

[0049] Example 8: Adsorption of nanoplastics in pre-cooked dishes by nanomagnetic beads 1. Experimental Methods This embodiment selects two common pre-cooked dishes: shredded pork with garlic sauce and Kung Pao chicken. The two dishes are processed according to the heating methods on the packaging: boiling for 6-7 minutes, microwaving for 3-4 minutes, and steaming for 10 minutes. After heat treatment, the nanoplastics in the pre-cooked dishes are extracted according to the processing methods for solid food, and then adsorbed using nano-magnetic beads.

[0050] 2. Experimental Results Plastic packaging can release nanoplastics after heat treatment, which can then migrate into food. Under different heating methods, pre-cooked dishes release a large amount of microplastics from the packaging and enter the food. After digesting Kung Pao Chicken and Shredded Pork with Garlic Sauce heated by boiling, microwaving, and steaming respectively, the nanoplastics were adsorbed using magnetic nanobeads. The results were as follows... Figure 7 As shown, the adsorption of nanoplastics in the above six pre-prepared food samples by the nanomagnetic beads can all reach more than 80%.

[0051] Example 9: Adsorption of nanoplastics released from food packaging materials by nanomagnetic beads 1. Experimental Methods In this embodiment, three commonly used materials—disposable lunch boxes, paper cups, and plastic wrap—were selected as research objects. 100 mL of distilled water heated to 15°C, 40°C, 60°C, 80°C, and 100°C, respectively, was used to heat-treat each material for 30 min. After treatment, the distilled water was transferred to pre-cleaned Erlenmeyer flasks, and 5 mg of magnetic nanoparticles were added. The flasks were incubated at 37°C on a shaker for 180 s. After adsorption, magnetic separation was performed, and the supernatant was collected. The supernatant was then filtered through a 0.22 μm microporous membrane. The membrane was observed under a microscope, and five regions were selected to count the nanoplastics. The adsorption rate of the magnetic nanoparticles on the nanoplastics was calculated using Formula 1.

[0052] Experimental results Disposable food packaging materials release a large amount of nanoplastics after heat treatment. This example first investigated the release of microplastics from three food packaging materials at different temperatures. The amount of nanoplastics released by the three packaging materials increased with increasing temperature. The nanomagnetic beads showed a high adsorption rate of over 80% for the nanoplastics released from the packaging materials treated at different temperatures (see...). Figure 8 ).

[0053] In summary, the magnetic nanobeads prepared by this invention can be adsorbed onto the surface of nanoplastics, achieving an extremely high adsorption rate in a very short time and exhibiting high adsorption efficiency. The optimal conditions for the adsorption of nanoplastics by the magnetic nanobeads are: an adsorption time of 150 seconds and a pH value of 9. Adsorption of nanoplastics in environmental samples, liquid foods, solid foods, semi-solid foods, pre-prepared dishes, and food packaging materials demonstrates that the magnetic nanobeads also have a good adsorption effect on nanoplastics in environmental and food samples.

[0054] Example 10: Preparation of fluorescent nanoplastics Experimental methods This experiment used purchased standard nanoplastic particles to prepare fluorescent nanoplastics. 1g of Nile Red dye was accurately weighed and added to 10mL of chloroform under light-protected conditions, and stored in the dark for later use. Different masses of standard nanoplastic particles were weighed and dispersed in 9mL of a 1:1 mixture of ultrapure water and dimethyl sulfoxide to achieve final nanoplastic concentrations of 0, 0.1, 0.2, 0.4, 0.8, and 1.0 μg / mL. 1mL of Nile Red dye solution was added to the nanoplastic solution at 75℃, heated for 30min, and then immediately placed in ice water to cool, thus obtaining the fluorescent nanoplastics (see...). Figure 9 ).

[0055] Experimental results Nanoplastics themselves do not carry fluorescence. In this embodiment, through certain treatments, the nanoplastics are made to carry fluorescence, which facilitates the detection of nanoplastics.

[0056] Example 11: Preparation of standard curves for fluorescent nanoplastics Experimental methods In this experiment, fluorescent nanoplastics of different concentrations obtained in Example 10 were placed in black ELISA plates, and absorbance values ​​were detected using an ELISA reader. The excitation wavelength was 543 nm and the emission wavelength was 620 nm. Standard curves were plotted based on the absorbance values ​​corresponding to different concentrations.

[0057] Experimental results The absorbance values ​​of fluorescent nanoplastics varied at different concentrations, and increased with increasing concentration. A standard curve was plotted based on the results obtained from the microplate reader, showing a positive correlation between the concentration of the fluorescent nanoplastics and the absorbance value (see...). Figure 10 ).

[0058] Rapid detection of nanoplastics in samples Experimental methods Under an applied magnetic field, the magnetic nanobeads can rapidly adsorb nanoplastics from the sample. Then, the pH of the complex of the magnetic nanobeads and nanoplastics causes the nanoplastics to desorb from the magnetic nanobeads. The desorbed nanoplastics are dispersed in 9 mL of a mixture of ultrapure water and dimethyl sulfoxide (1:1). 1 mL of Nile red dye solution is added to the nanoplastic solution at 75 °C, and the mixture is heated for 30 min. Then, it is immediately placed in ice water to cool. The resulting sample is placed in an ELISA plate, and the absorbance value is detected using an ELISA reader with an excitation wavelength of 543 nm and an emission wavelength of 620 nm.

[0059] Experimental results Disposable food packaging materials release a large amount of microplastics after heat treatment. In this embodiment, nanomagnetic beads are first used to adsorb the nanoplastics in the sample, then fluorescent labeling is performed, and finally the absorbance value is detected by an enzyme-linked immunosorbent assay (ELISA) reader. The obtained absorbance value is substituted into the standard curve to obtain the concentration of nanoplastics in the sample.

[0060] In summary, the method for rapid detection of nanoplastics using self-made magnetic nanobeads proposed in this invention can detect nanoplastics in food samples in a very short time; however, the preparation of fluorescent nanoplastics must be carried out under light-protected conditions. Based on the adsorption effect of the aforementioned magnetic nanobeads, this method is applicable to the detection of nanoplastics in environmental samples, liquid foods, solid foods, semi-solid foods, pre-prepared dishes, and food packaging materials.

[0061] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above... The description should not be considered a limitation of the invention. Various modifications and substitutions to the invention will be apparent to those skilled in the art after reading the foregoing. Therefore, the scope of protection of the invention should be defined by the appended claims.

Claims

1. A rapid detection method for nanoplastics in food and environmental samples based on self-made nanomagnetic beads, characterized in that, Includes the following steps: S1. Preparation of nanomagnetic beads: (1) Add 1.5g FeCl2, 4.05g FeCl3 and 75mL ultrapure water to a three-necked flask, and react with a magnetic stirrer at 70-80℃ in a 37.5mL ammonia environment for 50-80min. Nitrogen gas is introduced for protection at a flow rate of 0.5g / min. After the solution turns deep orange, add 0.5mL oleic acid and continue to react at 60-70℃ for 40-50min. (2) Perform magnetic separation on the reaction mixture. First wash with 100 mL of deionized water 2-3 times, then wash with 80 mL of anhydrous ethanol once, and then perform ultrasonic treatment for 5-10 min before magnetic separation again. (3) Add 30 mL of 0.1 g / mL 2-(n-morpholine) ethanesulfonic acid-hydrate solution sequentially, and sonicate at 30 °C for 20 min with an ultrasonic power of 0.5 W / cm². 2 After magnetic separation, 40 mL of 0.1 g / mL 2-(n-morpholine)ethanesulfonic acid-hydrate solution was added, and the mixture was sonicated at 30 °C for 30 min. Then, 70 mL of 0.04 g / mL carbodiimide solution was added, and the mixture was incubated on a shaker at 25 °C for 40 min. After freeze-drying, nano-magnetic bead powder was obtained. S2. Preparation of standard curves for fluorescent nanoplastics: (1) Dissolve 1g of Nile Red dye in 10mL of chloroform under light-protected conditions to prepare Nile Red dye solution and store it in the dark. (2) Weigh different masses of standard nanoplastics and disperse them in a 9 mL mixture of ultrapure water and dimethyl sulfoxide to prepare nanoplastic solutions with concentrations of 0, 0.1, 0.2, 0.4 and 0.8 μg / mL; (3) At 75°C, add 1 mL of Nile Red dye solution to each concentration of nanoplastic solution, heat for 30 min and then immediately cool with ice water to obtain fluorescent nanoplastics; (4) Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance of fluorescent nanoplastics. The excitation wavelength is 543 nm and the emission wavelength is 620 nm. Plot a standard curve based on the relationship between absorbance and concentration. S3. Detection of nanoplastics in samples: (1) Add the nanomagnetic beads obtained in step S1 to food or environmental samples and incubate them at 37°C and pH 3-12 for 30-180s. The adsorption of nanoplastics by the nanomagnetic beads is achieved by applying an external magnetic field. (2) Adjust the pH value of the nanomagnetic beads and nanoplastic composite to desorb the nanoplastic; when pH>10, the desorption effect is significant, and the pH can be adjusted by slowly adding 25% concentrated ammonia or 1mol / L sodium carbonate solution; collect the desorbed nanoplastic and perform fluorescent labeling according to the method in (2) and (3) of step S2; (3) Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance value of the labeled sample, and substitute it into the standard curve to calculate the concentration of nanoplastics in the sample.

2. The method according to claim 1, characterized in that, In step S2 (3), the heating method is a constant temperature water bath, and the enzyme-linked immunosorbent assay (ELISA) test must be completed within 30 minutes after cooling.

3. The method according to claim 1, characterized in that, In step S3(1), the mass-to-volume ratio of the nanomagnetic beads to the sample is 1:2.

5.

4. The method according to claim 1, characterized in that, In step S3, the required concentration of Nile Red dye for the preparation of the fluorescent nanoplastics is 10 μg / mL.

5. The method according to claim 1, characterized in that, The ratio of ultrapure water to dimethyl sulfoxide in step S2 (2) is 1:

1.

6. The method according to claim 1, characterized in that, The food samples include liquid food, solid food, semi-solid food, and pre-prepared dishes; the environmental samples include soil and water.

7. The method according to claim 1, characterized in that, The nanomagnetic beads obtained in step S1 have a particle size ≤30nm, are regularly spherical, and have amino groups on their surface.

8. The method according to claim 1, characterized in that, In step S3, the adsorption rate of the nanomagnetic beads on the nanoplastic is ≥80%.

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