14C and fluorescent molecule double-labeled polystyrene nanoparticles as well as preparation method and application thereof

Abstract

The invention relates to the technical field of environmental pollutant detection, and discloses 14C and fluorescent molecule double-labeled polystyrene nanoparticles as well as a preparation method and application thereof, the polystyrene nanoparticles have the following structural formula, * C represents 14C, the range of m is 30-100, the range of n is 10-30, and m: n = (3-10): 1. The preparation method comprises the following steps: preparing a 14C-styrene monomer by using 14C-containing methyl triphenyl phosphine iodide as a raw material, and polymerizing the 14C-styrene monomer with N-3-butenyl-7-nitro-2, 1, 3-benzofuran-4-amine to obtain the polystyrene nanoparticles. The PS-NPs marked by the radioisotope 14C and the fluorescent molecule can be traced by means of the radioisotope, the trace amount is accurate, and the PS-NPs are irreplaceable, so that the micro-plastic can be subjected to fluorescence imaging, and the micro-plastic nano-particles can be visually and accurately positioned. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of environmental pollutant detection, and in particular to a 14 Polystyrene nanoparticles dual-labeled with C and fluorescent molecules, and preparation method and application thereof. Background Art

[0002] PS is the most common plastic polymer in the environment and is widely distributed in human daily life. Currently, a large number of studies have used PS as a model compound to simulate the environmental behavior of microplastics in organisms in marine and terrestrial ecosystems.

[0003] For example, in terms of environmental factors, studies have found that high salinity can promote the further aggregation of PS-NH2 and unmodified polystyrene MPs / NPs, while the presence of negative charges on the surface of PS-COOH increases its transport rate in the environment. In plants, many studies have shown that PS-MPs / NPs can accumulate in seeds, stems, and other places, and have important effects on plant physiology and biochemistry. For example, in wheat seed germination, PS-MPs / NPs affect root growth rate and plant biomass, while hindering the transport and accumulation of essential nutrients such as iron, manganese, copper, and zinc in wheat, and disrupting gene expression and ion transport. In addition, exposure to PS-NPs can cause changes in the growth of plant root structure, leading to interruptions in the biosynthesis of plant root-related gene expression, phenylpropanoid biosynthesis, and fatty acid metabolism.

[0004] In terms of animal uptake, Setala et al. reported that fluorescent polystyrene (PS) microspheres (10 μm) can be ingested by zooplankton shrimp and spread through planktonic nutrition. Recently, Buwono et al. demonstrated that the abundance of MPs (0.0001-1.0 mm) has direct and indirect biological effects on oxidative damage in shrimp gills and digestive tract. In mammalian studies, An et al. found that polystyrene particles can reduce GSH-Px, CAT and SOD in the ovaries of fed female rats, increase MDA activity, and cause reproductive toxicity in mice.

[0005] However, there is a lack of research on the quantitative distribution of PS-MPs / NPs in plants and animals, and the physiological, biochemical and cytotoxic effects related to micro-nano plastics still need to be further studied. In order to accurately evaluate the transport mechanism of micro-nano plastics in plant tissues and organs, quantitative description of their concentration in plant tissues becomes the key.

[0006] The fluorescent groups in most fluorescent nanoplastics reported so far are physically adsorbed on the polymer surface, and their stability is poor, so they are easily detached due to factors such as dye concentration and ambient temperature. CN116606642A discloses a long-lasting fluorescent labeling nanoplastic and its preparation method and application, wherein the nanoplastic is a core-shell structure, i.e., it is composed of an intermediate core and an outer shell; the intermediate core is a biological window excited near-infrared long-lasting nanoparticle, and the outer shell is a polystyrene plastic; the biological window excited near-infrared long-lasting nanoparticle is synthesized with the help of a mesoporous silica template.

[0007] CN103172941A discloses a polystyrene fluorescent nanoparticle and its preparation and application. The particle is a polystyrene fluorescent nanoparticle with a fluorescent substance, wherein the fluorescent substance is a chelate formed by the reaction of a rare earth element with a β-diketone substance and tri-n-octylphosphine oxide. The preparation method comprises: 1) synthesizing polystyrene nanoparticles by emulsion polymerization, seed emulsion polymerization or soap-free emulsion polymerization; 2) adding polystyrene nanoparticles swollen with an organic solvent to an organic solvent in which the fluorescent substance is dissolved, and removing the organic solvent after the fluorescent substance diffuses into the interior of the polystyrene nanoparticles.

[0008] However, due to the high or low hydrophilicity or hydrophobicity exhibited by different MPs / NPs, staining with a single dye usually does not result in staining of all types of MPs / NPs. Therefore, some samples are easily overlooked during the quantification process. For some samples in animals and plants, organic tissue residues caused by incomplete digestion during the sample processing process will be co-stained with MPs, which will cause deviations in the MPs count in the sample. Summary of the invention

[0009] The present invention aims to solve the problems of poor stability of fluorescent labeling and insufficient quantitative accuracy of PS-NPs microplastics and provides a radioisotope 14 Polystyrene nanoparticles simultaneously labeled with C and the fluorescent molecule NBD-A can achieve precise positioning and quantification with the help of radioactive isotopes.

[0010] To achieve the above object, the technical solution adopted by the present invention is:

[0011] A sort of 14 C and fluorescent molecules dual-labeled polystyrene nanoparticles, the polystyrene nanoparticles having the following structural formula:

[0012]

[0013] Among them, *C represents 14 C, m ranges from 30-100, n ranges from 10-30, m:n=3-10:1.

[0014] The particle size of the polystyrene nanoparticles is 80-120 nm, and the specific activity is 40-60 μCi / mg;

[0015] The number average molecular weight of the polystyrene nanoparticles is 7500-8000 g / mol, and the molecular weight distribution coefficient is 1.6-2.0.

[0016] The polystyrene nanoparticles have an excitation wavelength of 310-320 nm and an emission wavelength of 625-635 nm.

[0017] The present invention also provides the 14 The method for preparing polystyrene nanoparticles double-labeled with C and fluorescent molecules comprises the following steps:

[0018] Step 1: 14 C-methyl triphenylphosphine iodide solution was added dropwise with n-butyl lithium solution for reduction reaction, and then benzaldehyde was added dropwise to the reaction solution for reaction, and the product was purified by extraction, distillation and column chromatography separation to obtain 14 C-styrene monomer;

[0019] Step 2, synthesizing N-3-butenyl-7-nitro-2,1,3-benzofuran-4-amine by reacting 4-butene-1-amine and 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) under the catalysis of N,N-diisopropylethylamine (DIPEA);

[0020] Step 3: 14 C-styrene, non-labeled styrene and N-3-butenyl-7-nitro-2,1,3-benzofuran-4-amine are mixed and dispersed, and then a potassium persulfate aqueous solution is added dropwise for polymerization reaction, and the product is purified to obtain the polystyrene nanoparticles.

[0021] Preferably, in step 1 14 Benzaldehyde is easily formed during the purification of C-styrene monomer. In the distillation process, inert gas is used to replace the air in the device. In step 3, since the synthesis process of radioactive polymers is slower than that of non-radiolabeled compounds, the reaction time should be appropriately extended.

[0022] In step 1, the reduction reaction temperature is -5 to 5°C, and the reduction reaction is performed for 0.5 to 2 hours;

[0023] The temperature is -5 to 5°C when benzaldehyde is added dropwise, and the reaction is carried out at room temperature for 10 to 24 hours after the addition is completed.

[0024] In step 1 14 The molar ratio of C-methyltriphenylphosphine iodide and n-butyl lithium is 1:0.8-1.2; benzaldehyde and 14 The molar ratio of C-methyltriphenylphosphonium iodide is 1:0.8-1.2.

[0025] In step 2, the reaction temperature is room temperature and the reaction time is 12h-48h;

[0026] The molar ratio of 4-butene-1-amine, 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole and N,N-diisopropylethylamine is 1:(3-5):(30-35). Step 3 is carried out under the protection of inert gas; 14 The molar ratio of C-styrene to non-labeled styrene is 1:3-10; the molar amount of N-3-butenyl-7-nitro-2,1,3-benzofuran-4-amine is 14 The total molar ratio of C-styrene and non-labeled styrene was 1:0.8-1.2;

[0027] In step 3, the polymerization reaction is carried out at 60-80° C. and the reaction time is 10-30 h.

[0028] The present invention also provides the 14 Application of polystyrene nanoparticles dual-labeled with C and fluorescent molecules in the detection of nano-microplastic pollutants.

[0029] The radioactive isotopes prepared in this patent 14 PS-NPs labeled with C and fluorescent molecule NBD-A can be used for precise quantitative research on the absorption, transport and enrichment of nanoplastic particles in organisms by virtue of the radioactive isotope tracing, trace accuracy and irreplaceable characteristics; at the same time, the fluorescent properties of microplastics themselves can be used to perform fluorescent imaging of microplastics and intuitively locate microplastic nanoparticles.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] Compared with the currently commercialized PS particles that are fluorescently labeled by physical methods, the fluorescent nanoplastics labeled by chemical methods in this patent are more chemically stable, and can prevent the fluorescent molecules from falling off during the long-life research process and truly reflect the behavior of the microplastics themselves. However, in a comprehensive comparison, the quantification of microplastics by radioisotope labeling has the advantages of high sensitivity and trace accuracy, and is an irreplaceable and powerful tool for studying the quantitative distribution of microplastics in biological tissues at environmental levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 for 14 HPLC-HRMS mass spectrum characterization of C-styrene.

[0033] Figure 2 For NBD-A 1 HNMR spectrum (500 MHz, CCl3D).

[0034] Figure 3This is the HPLC-HRMS spectrum of NBD-A.

[0035] Figure 4 for 14 SEM image of C / NBD-A dual-labeled polystyrene nanoparticles.

[0036] Figure 5 for 14 Fluorescence spectra of C / NBD-A dual-labeled polystyrene nanoparticles, a is the original sample, b is the sample after natural weathering for 15 days and washing with ethanol three times. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiment. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Those skilled in the art can make modifications or equivalent substitutions based on the understanding of the technical scheme of the present invention without departing from the spirit and scope of the technical scheme of the present invention, and all should be included in the protection scope of the present invention.

[0038] The raw materials used in the following specific embodiments are all purchased from the market.

[0039] Example 1

[0040] Step 1, Preparation 14 C-styrene monomer: 505 mg of methyltriphenylphosphine iodide [ 14 C] and 5 ml of anhydrous tetrahydrofuran. Stir evenly at 0°C with a magnetic stirrer, and add 2M n-butyl lithium in hexane solution (0.625 ml, 1.25 mmol) dropwise to react. The resulting mixture was stirred at room temperature for 1 hour to obtain an orange suspension. The mixture was then cooled to 0°C again, and 132 mg (1.25 mmol) of benzaldehyde solution was added dropwise to react. The reaction was then stirred at room temperature for 15 hours. After the reaction, thin layer chromatography (10:1 hexane / ethyl acetate) was performed, and most of the methyltriphenylphosphine iodide [ 14 C] conversion. After the reaction, 2.0 mL of water was used to quench the reaction at 0°C and 3x 5.0 mL of ether was used for extraction. The extracted organic solution was washed with 5.0 mL of saturated NaHCO3 solution, dried over anhydrous Na2SO4, and rotary evaporated and concentrated to obtain a crude product. The crude product was eluted by 20:1 n-hexane / ether silica gel chromatography to obtain about 60 mg (~50%) of the desired product. The reaction formula is as follows:

[0041]

[0042] like Figure 1As shown, the results of HPLC-HRMS characterization showed that the molecular weight of the target compound was 14 C-Molecular ion peak obtained by combining a proton with styrene monomer [M+H] + Its mass number is 106.0812, which is close to 106.0612 of the target molecule, and the single peak in liquid chromatography indicates that the chemical purity of the obtained product is high.

[0043] Step 2: Prepare the fluorescent molecule NBD-A monomer, add 650 mg of NBD-Cl, 242.0 mg of 4-butene-1-amine, 2.5 mL of DIPEA and 11.0 mL of methanol solvent into a three-necked flask, stir and react at room temperature, and use TLC thin-layer chromatography to track the reaction process until the raw material NBD-Cl reacts completely. After the reaction is completed, ethyl acetate and cyclohexane volume ratio (1:1 to 1:3) are used as eluents, and separated and purified by silica gel column chromatography to obtain 0.30 g of solid product with a yield of 40%. The reaction formula is as follows:

[0044]

[0045] like Figure 2 As shown, 1 The results of H-NMR spectrum test showed that the ratio of the chemical shift of protons and the integrated area of ​​the corresponding proton nuclear magnetic vibration peaks in the synthesized compound was 1:1:2:2:2:2, which was consistent with the organic molecular structure of NBD-A. Further characterization and analysis by HPLC-HRMS showed that Figure 3 As shown, the results show that the m / z of NBD-A is 233.0668, while the accurate molecular mass of the target product is 234.0681, with an error within ±0.05, which is consistent with the target molecular weight of NBD-A.

[0046] Step 3: Add deionized water to a three-necked flask equipped with a stirrer, condenser, and nitrogen tube, and fill with nitrogen for 30 minutes to more completely remove oxygen from the water. Keep the speed at 180 rpm and drop 0.2 g of 14 C-styrene and non-labeled styrene monomer (the molar ratio of the two is 1:5), 3mg of fluorescent molecule NBD-A are added to a three-necked flask, stirred and dispersed at room temperature for 30min, and then 10.0mL of an aqueous solution containing 23.0mg of K2S2O8 is added dropwise for reaction. After the addition is completed, the reaction system is heated to 70°C and the reaction is continued for 24 hours. Nitrogen is continuously introduced during the reaction to prevent oxidation of the product. After the reaction is completed, it is washed with ethanol 3-5 times, centrifuged at 12000rpm / 60min and dried to obtain a particle size of 80-120nm, a specific activity of 49μCi / mg, and a mass yield of 3.6%.

[0047] Prepared14 SEM images of C / NBD-A dual-labeled polystyrene nanoparticles Figure 4 As shown, the microspheres have uniform particle size and smooth surface, with a size distribution of about 80-120 nm, which has not changed significantly compared with the particle size of non-labeled PS-NPs.

[0048] like Figure 5 As shown in the fluorescence spectrum experimental results, the synthesized 14 The excitation wavelength of C / NBD-A dual-labeled polystyrene is 314nm, and the emission wavelength is 630nm; the excitation wavelength of Eu-PS-NPs is 314nm, and the emission wavelength is 631nm, which also shows red fluorescence. After being weathered in the environment for 15 days and washed with ethanol many times, the excitation and emission spectra were still stable after re-experimental measurement, indicating that the chemically complexed fluorescent group is embedded in the surface of PS-NPs and has stable chemical properties.

[0049] As shown in Table 1, the gel chromatography GPC analysis results show that the synthesized radioisotope and fluorescent molecule double labeled 14 The weight average molecular weight (Mw), number average molecular weight (Mn), Z average molecular weight (Mz) and molecular weight distribution (PDI) of C / NBD-A-PS-NPs polystyrene particles are 10848, 7813, 19054 and 1.83, respectively, indicating that the molecular weight distribution of the prepared polystyrene microspheres is relatively wide.

[0050] Table 1. Synthesis 14 C / NBD-A dual-labeled polystyrene particle gel chromatography (GPC) molecular weight test results

[0051] category Mw(g / mol) Mn(g / mol) Mz(g / mol) PDI <![CDATA[ 14 C-Fl-PS-NPs]]> 10848 7813 19054 1.83

Claims

1. A 14 C and fluorescent molecule dual-labeled polystyrene nanoparticles, characterized in that: The polystyrene nanoparticles have the following structural formula: Among them, *C represents 14 C, m ranges from 30-100, n ranges from 10-30, m:n=3-10:

1.

2. according to claim 1 14 C and fluorescent molecule dual-labeled polystyrene nanoparticles, characterized in that: The particle size of the polystyrene nanoparticles is 80-120 nm, and the specific activity is 40-60 μCi / mg.

3. According to claim 1 14 C and fluorescent molecule dual-labeled polystyrene nanoparticles, characterized in that: The number average molecular weight of the polystyrene nanoparticles is 7500-8000 g / mol, and the molecular weight distribution coefficient is 1.6-2.

0.

4. according to claim 1 14 C and fluorescent molecule dual-labeled polystyrene nanoparticles, characterized in that: The polystyrene nanoparticles have an excitation wavelength of 310-320 nm and an emission wavelength of 625-635 nm.

5. The method according to any one of claims 1 to 4 14 The method for preparing polystyrene nanoparticles dual-labeled with C and fluorescent molecules is characterized in that: Includes steps: Step 1: 14 C-methyl triphenylphosphine iodide solution was added dropwise with n-butyl lithium solution for reduction reaction, and then benzaldehyde was added dropwise to the reaction solution for reaction, and the product was purified by extraction, distillation and column chromatography separation to obtain 14 C-styrene monomer; Step 2, synthesizing N-3-butenyl-7-nitro-2,1,3-benzofuran-4-amine by reacting 4-butene-1-amine and 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole under the catalysis of N,N-diisopropylethylamine; Step 3: 14 C-styrene, non-labeled styrene and N-3-butenyl-7-nitro-2,1,3-benzofuran-4-amine are mixed and dispersed, and then a potassium persulfate aqueous solution is added dropwise for polymerization reaction, and the product is purified to obtain the polystyrene nanoparticles.

6. According to claim 5 14 The method for preparing polystyrene nanoparticles dual-labeled with C and fluorescent molecules is characterized in that: In step 1, the reduction reaction temperature is -5 to 5°C, and the reduction reaction is performed for 0.5 to 2 hours; The temperature is -5 to 5°C when benzaldehyde is added dropwise, and the reaction is carried out at room temperature for 10 to 24 hours after the addition is completed.

7. According to claim 5 14 The method for preparing polystyrene nanoparticles dual-labeled with C and fluorescent molecules is characterized in that: In step 1 14 The molar ratio of C-methyltriphenylphosphine iodide and n-butyl lithium is 1:0.8-1.2; benzaldehyde and 14 The molar ratio of C-methyltriphenylphosphonium iodide is 1:0.8-1.

2.

8. According to claim 5 14 The method for preparing polystyrene nanoparticles dual-labeled with C and fluorescent molecules is characterized in that: In step 2, the reaction temperature is room temperature and the reaction time is 12h-48h; The molar ratio of 4-butene-1-amine, 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole and N,N-diisopropylethylamine is 1:(3-5):(30-35).

9. According to claim 5 14 The method for preparing polystyrene nanoparticles dual-labeled with C and fluorescent molecules is characterized in that: Step 3 is carried out under the protection of inert gas; 14 The molar ratio of C-styrene to non-labeled styrene is 1:3-10; the molar amount of N-3-butenyl-7-nitro-2,1,3-benzofuran-4-amine is 14 The total molar ratio of C-styrene and non-labeled styrene was 1:0.8-1.2; In step 3, the polymerization reaction is carried out at 60-80° C. and the reaction time is 10-30 h.

10. The method according to any one of claims 1 to 4 14 Application of polystyrene nanoparticles dual-labeled with C and fluorescent molecules in the detection of nano-microplastic pollutants.

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