A 14 C and fluorescent molecule double-labeled polystyrene nanoparticles, preparation method and application thereof
By using polystyrene nanoparticles double-labeled with 14C and the fluorescent molecule NBD-A, the problem of poor stability of fluorescent labeling was solved, enabling precise localization and quantitative study of microplastics in organisms, and providing a highly sensitive and trace-precise research tool.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ZHEJIANG UNIV
- Filing Date
- 2024-09-29
- Publication Date
- 2026-05-19
AI Technical Summary
In existing technologies, the fluorescent labeling stability of polystyrene microplastics is poor, resulting in insufficient quantitative accuracy. Furthermore, it is difficult to accurately stain all types of micro- and nano-plastics with a single dye, affecting the accuracy of quantitative studies in animals and plants.
Polystyrene nanoparticles were double-labeled with the radioactive isotope 14C and the fluorescent molecule NBD-A. The fluorescent molecule was stably embedded into the surface of the polystyrene nanoparticles through a chemical method, and the radioactive isotope was used to achieve precise localization and quantification.
It achieves improved chemical stability of fluorescent labels, preventing the shedding of fluorescent molecules in long-term studies, and provides a highly sensitive and precise microplastic quantification tool suitable for the accurate localization and quantitative distribution of microplastics in vivo.
Smart Images
Figure CN119219820B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of environmental pollutant detection technology, specifically to a... 14 Polystyrene nanoparticles double-labeled with C and fluorescent molecules, their preparation methods and applications. Background Technology
[0002] PS, as the most common plastic polymer in the environment, is widely distributed in everyday life where humans can come into contact with it. Currently, a large number of studies use PS as a model compound to simulate the environmental behavior of microplastics in organisms in marine and terrestrial ecosystems.
[0003] For example, regarding environmental factors, studies have found that high salinity can promote the further accumulation of PS-NH2 and unmodified polystyrene MPs / NPs, while the presence of negative surface charges on PS-COOH increases their transport rate in the environment. In plants, numerous studies have shown that PS-MPs / NPs can accumulate in seeds, stems, and other locations, significantly impacting plant physiology and biochemistry. For instance, during wheat seed germination, PS-MPs / NPs affect root growth rate and plant biomass, while also hindering the transport and accumulation of essential nutrients such as iron, manganese, copper, and zinc, disrupting gene expression and ion transport. Furthermore, PS-NP exposure alters the growth of plant root structures, leading to the interruption of root-related gene expression, phenylpropanoid biosynthesis, and fatty acid metabolism.
[0004] Regarding animal uptake, Setala et al. reported that fluorescent polystyrene (PS) microspheres (10 μm) can be ingested by zooplankton shrimp and dispersed via planktonic nutrition. More recently, Buwono et al. demonstrated that the abundance of MPs (0.0001–1.0 mm) has direct and indirect biological effects on oxidative damage to shrimp gills and digestive tracts. In mammalian studies, An et al. found that polystyrene particles reduced GSH-Px, CAT, and SOD levels and increased MDA activity in the ovaries of fed female rats, causing reproductive toxicity in mice.
[0005] However, quantitative studies on the distribution of PS-MPs / NPs in plants and animals are still relatively scarce, and the physiological, biochemical, and cytotoxic effects related to micro- and nano-plastics require further investigation. Quantitatively describing their concentration in plant tissues is crucial for accurately assessing the transport mechanisms of micro- and nano-plastics in plant tissues and organs.
[0006] Most fluorescent nanoplastics reported to date have fluorescent groups that are adsorbed onto the polymer surface through physical action, resulting in poor stability and easy detachment due to factors such as dye concentration and ambient temperature. CN116606642A discloses a long-afterglow fluorescent labeled nanoplastic, its preparation method, and its application. The nanoplastic has a core-shell structure, consisting of an intermediate core and an outer shell. The intermediate core is a bio-window-excited near-infrared long-afterglow nanoparticle, and the outer shell is polystyrene plastic. The bio-window-excited near-infrared long-afterglow nanoparticle is synthesized using a mesoporous silica template.
[0007] CN103172941A discloses a polystyrene fluorescent nanoparticle and its preparation and application. The particle is a polystyrene fluorescent nanoparticle with fluorescent material, wherein the fluorescent material is a chelate formed by the reaction of rare earth elements with β-diketones and tri-n-octylphosphine oxide. The preparation method includes: 1) synthesizing polystyrene nanoparticles by emulsion polymerization, seed emulsion polymerization or soap-free emulsion polymerization; 2) adding polystyrene nanoparticles swollen by organic solvent to an organic solvent containing fluorescent material, and after the fluorescent material diffuses into the interior of the polystyrene nanoparticles, the organic solvent is removed.
[0008] However, due to the high or low hydrophilicity or hydrophobicity of 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 quantification. For some animal and plant samples, organic tissue residues caused by incomplete digestion during the processing may co-stain with MPs, which may cause deviations in the MP count in the sample. Summary of the Invention
[0009] This invention addresses the problems of poor fluorescent labeling stability and insufficient quantitative accuracy in PS-NPs microplastics by providing a radioactive isotope... 14 Polystyrene nanoparticles simultaneously labeled with C and the fluorescent molecule NBD-A can be precisely located and quantified using radioactive isotopes.
[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0011] A sort of 14 Polystyrene nanoparticles double-labeled with C and fluorescent molecules, wherein the polystyrene nanoparticles have the following structural formula:
[0012]
[0013] Where *C represents 14 C, m ranges from 30 to 100, n ranges from 10 to 30, and m:n = 3-10:1.
[0014] The polystyrene nanoparticles have a particle size of 80-120 nm and a specific activity of 40-60 μCi / mg;
[0015] The polystyrene nanoparticles have a number-average molecular weight of 7500-8000 g / mol and a molecular weight distribution coefficient of 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 aforementioned 14 The method for preparing polystyrene nanoparticles double-labeled with C and fluorescent molecules includes the following steps:
[0018] Step 1, to contain 14 The product was obtained by dropwise reduction of C-methyltriphenylphosphine iodide solution with n-butyllithium solution, followed by dropwise addition of benzaldehyde. The product was purified by extraction, distillation, and column chromatography. 14 C-Styrene monomer;
[0019] Step 2: 4-Buten-1-amine and 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) are synthesized into N-3-butenyl-7-nitro-2,1,3-benzofuran-4-amine under the catalysis of N,N-diisopropylethylamine (DIPEA);
[0020] Step 3, 14 C-styrene, unlabeled styrene, and N-3-butenyl-7-nitro-2,1,3-benzofuran-4-amine were mixed and dispersed, and then a potassium persulfate aqueous solution was added dropwise for polymerization. The product was purified to obtain the polystyrene nanoparticles.
[0021] Preferably, in step 1 14 Benzaldehyde is easily formed during the purification of C-styrene monomer. During the purification process, inert gas is used to replace the air in the device during distillation. In step 3, since the synthesis 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℃, and the reduction reaction time is 0.5 to 2 hours.
[0023] The temperature during the addition of benzaldehyde is -5 to 5℃, and the reaction is carried out at room temperature for 10-24 hours after the addition is completed.
[0024] In step 1 14 The molar ratio of C-methyltriphenylphosphine iodide to n-butyllithium is 1:0.8-1.2; benzaldehyde and 14 The molar ratio of C-methyltriphenylphosphine 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-buten-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 an inert gas atmosphere. 14 The molar ratio of C-styrene to unlabeled 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 to unlabeled styrene is 1:0.8-1.2;
[0027] In step 3, the polymerization reaction is carried out at 60-80℃ for 10-30 hours.
[0028] The present invention also provides the aforementioned 14 Application of polystyrene nanoparticles double-labeled with C and fluorescent molecules in the detection of nanoplastic pollutants.
[0029] The radioactive isotopes prepared in this patent 14 PS-NPs labeled with C and the fluorescent molecule NBD-A can be used to accurately quantify the absorption, transport, and accumulation of nanoplastic particles in organisms by leveraging the radioactive isotope tracing, trace precision, and irreplaceable characteristics. At the same time, the fluorescence properties of microplastics themselves can be used to perform fluorescence imaging to directly and accurately locate microplastic nanoparticles.
[0030] Compared with the prior art, the present invention has the following beneficial effects:
[0031] Compared to commercially available PS particles labeled with fluorescent markers using physical methods, the fluorescent nanoplastics labeled using chemical methods in this patent exhibit stronger chemical stability. This prevents the shedding of fluorescent molecules during long-term studies, thus accurately reflecting the behavior of the microplastics themselves. However, considering all factors, the radioactive isotope labeling method for quantifying microplastics offers advantages such as high sensitivity and precise trace-level measurement, making it an irreplaceable and powerful tool for studying the quantitative distribution of micro- and nanoplastics in biological tissues at environmental levels. Attached Figure Description
[0032] Figure 1 for 14 HPLC-HRMS mass spectrometry characterization of C-styrene.
[0033] Figure 2 For NBD-A 1 HNMR spectrum (500MHz, 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 double-labeled polystyrene nanoparticles, a is the original sample, b is the sample after natural weathering for 15 days and washing with ethanol 3 times. Detailed Implementation
[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Modifications or equivalent substitutions made by those skilled in the art based on their understanding of the technical solutions of this invention, without departing from the spirit and scope of the invention, should be covered within the protection scope of this invention.
[0038] All raw materials used in the following specific implementation methods were purchased from the market.
[0039] Example 1
[0040] Step 1, Preparation 14 C-Styrene monomer: Add 505 mg of methyltriphenylphosphine iodide to a 25 ml three-necked flask under argon gas flow. 14 C] and 5 ml of anhydrous tetrahydrofuran. Stirred at 0°C with a magnetic stirrer until homogeneous, and then add 2 M n-butyllithium in hexane solution (0.625 ml, 1.25 mmol) dropwise. 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 added dropwise with 132 mg (1.25 mmol) benzaldehyde solution. The reaction was then stirred at room temperature for 15 h, followed by thin-layer chromatography (10:1 hexane / ethyl acetate). Most of the methyltriphenylphosphine iodide [ 14 [C] Conversion. The reaction was quenched with 2.0 mL of water at 0 °C, and extracted with 3 x 5.0 mL diethyl ether. The extracted organic liquid was washed with 5.0 mL of saturated NaHCO3 solution, dried over anhydrous Na2SO4, and concentrated by rotary evaporation to obtain the crude product. The crude product was eluted by silica gel chromatography with a 20:1 hexane / diethyl ether ratio to give approximately 60 mg (~50%) of the desired product. The reaction formula is as follows:
[0041]
[0042] like Figure 1As shown, the results of high-performance liquid chromatography-high-resolution mass spectrometry characterization indicate that the molecular weight of the target compound is [missing information]. 14 The molecular ion peak [M+H] obtained by the combination of a proton with the C-styrene monomer. + Its mass number is 106.0812, which is close to that of the target molecule, and the single peak in the liquid chromatography indicates that the obtained product has high chemical purity.
[0043] Step 2: Preparation of the fluorescent molecule NBD-A monomer. 650 mg of NBD-Cl, 242.0 mg of 4-buten-1-amine, 2.5 mL of DIPEA, and 11.0 mL of methanol were added to separate three-necked flasks and stirred at room temperature. The reaction was monitored using thin-layer chromatography (TLC) until the starting material NBD-Cl was completely reacted. After the reaction was complete, ethyl acetate and cyclohexane (volume ratio 1:1–1:3) were used as eluents, and the mixture was 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, via 1 1H-NMR spectroscopy results showed that the chemical shift of the protons and the corresponding integrated area ratio of the proton nuclear magnetic vibration peaks in the synthesized compound were 1:1:2:2:2:2, consistent with the organic molecular structure of NBD-A. Further HPLC-HRMS characterization analysis revealed... Figure 3 As shown, the results indicate that the m / z of NBD-A is 233.0668, while the precise molecular weight of the target product is 234.0681, with an error within ±0.05. This result 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. Purge with nitrogen for 30 minutes to remove oxygen from the water more completely. Add 0.2g of the solution dropwise while maintaining a speed of 180 rpm. 14 C-styrene and unlabeled styrene monomers (molar ratio 1:5), along with 3 mg of the fluorescent molecule NBD-A, were added to a three-necked flask. After stirring and dispersing at room temperature for 30 min, 10.0 mL of an aqueous solution containing 23.0 mg of K₂S₂O₈ was added dropwise. After the addition was complete, the reaction system was heated to 70 °C and the reaction was continued for 24 hours, with nitrogen continuously purging to prevent oxidation of the product. After the reaction was complete, the product was washed 3-5 times with ethanol, centrifuged at 12000 rpm for 60 min, and dried to obtain particles with a size of 80-120 nm, a specific activity of 49 μCi / mg, and a yield of 3.6%.
[0047] Prepared14 SEM images of C / NBD-A dual-labeled polystyrene nanoparticles are shown below. Figure 4 As shown, the microspheres have uniform particle size and smooth surface, with a size distribution of approximately 80-120 nm. Compared to unlabeled PS-NPs, the particle size has not changed significantly.
[0048] like Figure 5 As shown, the fluorescence spectroscopy results indicate that the synthesized 14 The C / NBD-A double-labeled polystyrene was excited at 314 nm and emitted at 630 nm; the Eu-PS-NPs were excited at 314 nm and emitted at 631 nm, also exhibiting red fluorescence. After being exposed to the environment for 15 days and washed multiple times with ethanol, the excitation and emission spectra remained stable upon re-testing, indicating that the chemically complexed fluorescent groups are embedded on the surface of the PS-NPs and are chemically stable.
[0049] As shown in Table 1, the results of gel permeation chromatography (GPC) analysis indicate that the synthesized radioisotope and fluorescent molecule dual-labeled... 14 The weight-average molecular weight (Mw), number-average molecular weight (Mn), Z-average molecular weight (Mz), and molecular weight distribution (PDI) of the C / NBD-A-PS-NPs polystyrene particles were 10848, 7813, 19054, and 1.83, respectively, indicating that the prepared polystyrene microspheres had a wide molecular weight distribution.
[0050] Table 1. Synthesis 14 C / NBD-A dual-labeled polystyrene particle gel chromatography (GPC) molecular weight determination 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 kind 14 Polystyrene nanoparticles dual-labeled with C and fluorescent molecules, characterized in that... The polystyrene nanoparticles have the following structural formula: Where *C represents 14 C, the polystyrene nanoparticles have a number-average molecular weight of 7500-8000 g / mol, m:n=3-10:1; the polystyrene nanoparticles have a molecular weight distribution coefficient of 1.6-2.0; The method for preparing the polystyrene nanoparticles includes the following steps: Step 1, to contain 14 The product was obtained by dropwise reduction of C-methyltriphenylphosphine iodide solution with n-butyllithium solution, followed by dropwise addition of benzaldehyde. The product was purified by extraction, distillation, and column chromatography. 14 C-Styrene monomer; Step 2: 4-Buten-1-amine and 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole are synthesized into N-3-butenyl-7-nitro-2,1,3-benzofuran-4-amine under the catalysis of N,N-diisopropylethylamine; Step 3, 14 C-styrene, unlabeled styrene, and N-3-butenyl-7-nitro-2,1,3-benzofuran-4-amine were mixed and dispersed, and then a potassium persulfate aqueous solution was added dropwise for polymerization. The product was purified to obtain the polystyrene nanoparticles.
2. As described in claim 1 14 Polystyrene nanoparticles dual-labeled with C and fluorescent molecules, characterized in that... The polystyrene nanoparticles have a particle size of 80-120 nm and a specific activity of 40-60 μCi / mg.
3. As described in claim 1 14 Polystyrene nanoparticles dual-labeled with C and fluorescent molecules, characterized in that... The polystyrene nanoparticles have an excitation wavelength of 310-320 nm and an emission wavelength of 625-635 nm.
4. The method according to any one of claims 1-3 14 A method for preparing polystyrene nanoparticles dual-labeled with C and fluorescent molecules, characterized in that, Including the following steps: Step 1, to contain 14 The product was obtained by dropwise reduction of C-methyltriphenylphosphine iodide solution with n-butyllithium solution, followed by dropwise addition of benzaldehyde. The product was purified by extraction, distillation, and column chromatography. 14 C-Styrene monomer; Step 2: 4-Buten-1-amine and 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole are synthesized into N-3-butenyl-7-nitro-2,1,3-benzofuran-4-amine under the catalysis of N,N-diisopropylethylamine; Step 3, 14 C-styrene, unlabeled styrene, and N-3-butenyl-7-nitro-2,1,3-benzofuran-4-amine were mixed and dispersed, and then a potassium persulfate aqueous solution was added dropwise for polymerization. The product was purified to obtain the polystyrene nanoparticles.
5. The method according to claim 4 14 A method for preparing polystyrene nanoparticles dual-labeled with C and fluorescent molecules, characterized in that, In step 1, the reduction reaction temperature is -5~5℃, and the reduction reaction time is 0.5-2h. The temperature during the addition of benzaldehyde is -5 to 5°C, and the reaction is carried out at room temperature for 10-24 hours after the addition is completed.
6. The method according to claim 4 14 A method for preparing polystyrene nanoparticles dual-labeled with C and fluorescent molecules, characterized in that, In step 1 14 The molar ratio of C-methyltriphenylphosphine iodide to n-butyllithium is 1:0.8-1.2; benzaldehyde and 14 The molar ratio of C-methyltriphenylphosphine iodide is 1:0.8-1.
2.
7. The method according to claim 4 14 A method for preparing polystyrene nanoparticles dual-labeled with C and fluorescent molecules, characterized in that, In step 2, the reaction temperature is room temperature and the reaction time is 12h-48h. The molar ratio of 4-buten-1-amine, 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole and N,N-diisopropylethylamine is 1:(3-5):(30-35).
8. The method according to claim 4 14 A method for preparing polystyrene nanoparticles dual-labeled with C and fluorescent molecules, characterized in that, Step 3 is carried out under inert gas protection; 14 The molar ratio of C-styrene to unlabeled 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 to unlabeled styrene is 1:0.8-1.2; In step 3, the polymerization reaction is carried out at 60-80℃ for 10-30 hours.
9. The claim 1-3 14 Application of polystyrene nanoparticles double-labeled with C and fluorescent molecules in the detection of nanoplastic pollutants.