Preparation method and application of core-shell structure europium complex-containing double-tracing polystyrene microspheres

Abstract

The invention relates to a preparation method and application of a core-shell structure europium complex-containing double-tracing polystyrene microsphere, and a double-tracing polystyrene microsphere material, namely a double-tracing EU (at) PS microsphere material, prepared by the preparation method disclosed by the invention can be used for preparing the europium complex-containing double-tracing polystyrene microsphere without changing the inherent characteristics of MNPs, especially the surface characteristics of the MNPs. Therefore, a feasible solution is provided for tracking, positioning and quantifying the MNPs in an actual environment, and the method is characterized by comprising the following steps: step 1, synthesizing a europium complex Eu (DBM) 3phen; and step 2, synthesizing EU (at) PS microspheres containing Eu (DBM) 3phen, a core particle in the microspheres is formed by dispersing Eu (DBM) 3phen in a copolymer, the copolymer is formed by copolymerizing styrene and methyl methacrylate, and a shell coating the core particle is polystyrene formed by polymerizing styrene.

Description

Technical Field

[0001] The invention relates to the technical field of europium-doped polystyrene microsphere EU@PS materials, in particular to a preparation method and application of double-tracer polystyrene microspheres containing europium complexes in a core-shell structure. Background Art

[0002] Microplastics (MPs) (size 1 μm to 5 mm) and nanoplastics (NPs) (size 1 nm to 1 μm) have been found in natural water bodies, soils, remote deserts, high altitude plateaus, deep seas and polar regions. They have become ubiquitous pollutants and have received widespread public attention. Micro-nanoplastics (MNPs) may pose a huge threat to the environment due to the increasing production of non-degradable polymers and plastics, unsustainable use, and inadequate or lack of waste management. To accurately assess the ecological risks of micro-nanoplastics, it is necessary to systematically understand the migration, transformation, fate and toxicity mechanisms of MNPs in complex environments and the impact of complex environmental conditions. However, there is a lack of suitable tracking materials to identify and quantify MNPs in environmental matrices.

[0003] One way to overcome these challenges and limitations is to label MNPs. Existing tracking labeling technologies are divided into four main types: fluorescent labeling, metal labeling, stable isotope labeling, and radioisotope labeling. The use of fluorescent dye labeling has the advantages of simplicity and low cost, and is the most widely used and widely used for the visualization and detection of MNPs. However, at the same time, fluorescent labeling will affect the changes in the surface properties of MNPs. Fluorescent dyes are prone to quenching, leaching, and are easily interfered by environmental background. Compared with fluorescent MNP labeling, metal labeling has lower size detection limits and lower background interference, and good stability. However, labeling metal nanoparticles, such as gold and platinum, is expensive, the labeling process is more complicated, and most of the labels are on the surface of MNPs, which may change the surface properties of MNPs. Stable isotope labeling is a very effective tool that can accurately track the fate of plastic-derived materials in the environment, but it is expensive and technically complex. Radioisotope labeling has radioactive risks, is strictly regulated, and has extremely high experimental requirements.

[0004] Therefore, a promising method to achieve a systematic understanding of the environmental risks of MNPs is to dope metal elements in MNPs; lanthanide complexes have obvious advantages. They can be identified and quantified by inductively coupled plasma mass spectrometry (ICP-MS), and they have a long luminescence lifetime, sharp emission spectrum and visible light excitation wavelength. On the other hand, a major challenge of labeling technology is to develop technologies that do not change the inherent properties of MNPs or only change minimal properties, especially surface properties. Core-shell labeling technology provides a feasible strategy. Its synthetic materials are highly stable and can achieve the core as a metal marker and the shell as a target substance. Therefore, this patent uses europium complexes (Eu(DMP)3phen, tris(1,3-diphenyl-1,3-propanedione)mono(1,10-phenanthroline)europium(III)) as the core to synthesize core-shell MNPs with dual tracers without changing the surface properties of the material, providing a feasible solution for tracking, positioning and quantification of MNPs in actual environments. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a method for preparing double-tracer polystyrene microspheres containing europium complexes with a core-shell structure and applications. The double-tracer polystyrene microsphere material prepared by the present invention, namely the double-tracer EU@PS microsphere material, can maintain the inherent properties of MNPs, especially the surface properties of MNPs, thereby providing a feasible solution for tracking, positioning and quantifying MNPs in actual environments.

[0006] The technical solution of the present invention is as follows:

[0007] A method for preparing double-traced polystyrene microspheres containing a core-shell structured europium complex, characterized in that it comprises the following steps:

[0008] Step 1, synthesizing the europium complex Eu(DBM)3phen;

[0009] Step 2, synthesizing EU@PS microspheres containing Eu(DBM)3phen, wherein the core particles in the microspheres are Eu(DBM)3phen dispersed in a copolymer, wherein the copolymer is formed by copolymerization of styrene and methyl methacrylate, and the shell covering the core particles is polystyrene formed by polymerization of styrene.

[0010] Step 1 includes the following steps:

[0011] Step 1.1, prepare solution A: dissolve 2 mM EuCl3·6H2O in 20 ml of anhydrous ethanol;

[0012] Step 1.2, prepare solution B: add 4 mM 1,10-phenanthroline to 20 ml of anhydrous ethanol containing 6 mM dibenzoylmethane DBM;

[0013] Step 1.3, heating solution A and solution B to 50-70° C. respectively under magnetic stirring, slowly adding solution A to solution B to react and synthesize Eu(DBM)3phen, the reaction time is 1-2 hours, and then cooling the reaction solution to room temperature;

[0014] Step 1.4, filtering the reaction solution to obtain Eu(DBM)3phen.

[0015] Step 2 includes the following steps:

[0016] Step 2.1, weigh 40-60 g of Eu(DMP)3phen and dissolve it in 0.4-0.6 ml of methyl methacrylate, and add 1.4-1.6 ml of styrene to obtain 2 ml of a mixed solution;

[0017] Step 2.2, add 37 mL of deionized water and 2 mL of the mixed solution into a 250 mL three-necked flask, and ultrasonicate for 10 min to make the solution in the flask evenly dispersed;

[0018] Step 2.3, the interfaces of the three-necked flask are respectively connected to a spherical condenser, a mechanical stirring paddle and a glass gas guide tube, the glass gas guide tube is connected to nitrogen, and the mechanical stirring speed is 200-300 rpm;

[0019] Step 2.4, after nitrogen is passed for 30 min, the constant temperature oil bath is heated to 70-80°C, 2 mL of 10 mg / mL potassium persulfate aqueous solution is added to the three-necked flask to react to generate a microsphere solution, and the reaction time is 10 h;

[0020] Step 2.5, after the microsphere solution is cooled to room temperature, it is transferred to a centrifuge tube and centrifuged at 5000-10000 rpm for 10 min using a high-speed centrifuge to remove the europium complex in the unreacted raw material in the system;

[0021] In step 2.6, an ultra-high speed centrifuge is used to centrifuge at a speed of 25000-30000 rpm for 15 min to remove styrene and methyl methacrylate in the unreacted raw materials in the system, and the lower solid material in the centrifuge tube, i.e., EU@PS microspheres, is collected. The collected EU@PS microspheres are added to deionized water and dispersed by ultrasound; after repeating the centrifugation procedure three times, the EU@PS microspheres are redispersed in deionized water and stored as samples in light-proof glass bottles and stored in a refrigerator at 4°C.

[0022] Step 1.3 includes the use of a magnetic stirring heater.

[0023] Step 1.3 includes adjusting the pH to pH 6 to pH 7 after solution A and solution B are completely mixed.

[0024] The mechanical stirring paddle in step 2.3 is made of polytetrafluoroethylene.

[0025] The double-tracer polystyrene microspheres obtained by the above-mentioned method for preparing double-tracer polystyrene microspheres containing a core-shell structure europium complex are used as nano-fluorescent EU@PS materials in biomedical research, characterized in that the nano-fluorescent EU@PS material is exposed to Chlamydomonas reinhardtii cells at a certain concentration gradient under room temperature; after the exposure, the cells are collected by centrifugation, photographed using a laser confocal microscope and a high-content imaging system, and compared with a blank control group of nano-MNPs materials without any labeling and unexposed materials to evaluate the visualization performance of the fluorescent nano-EU@PS material.

[0026] The double-tracer polystyrene microspheres obtained by the above-mentioned method for preparing double-tracer polystyrene microspheres containing a core-shell structure europium complex are used as nano-fluorescent EU@PS materials in biomedical research, characterized in that the nano-fluorescent EU@PS material is exposed to Daphnia magna at a certain concentration gradient under room temperature; after the exposure, photos are taken using a high-content imaging system, and compared with a blank control group of nano-MNPs materials without any labeling and unexposed materials to evaluate the visualization performance of the fluorescent nano-EU@PS material.

[0027] The double-tracer polystyrene microspheres obtained by the above-mentioned method for preparing double-tracer polystyrene microspheres containing core-shell structure europium complexes are used as nano-fluorescent EU@PS materials in biomedical research, characterized in that the nano-fluorescent EU@PS material is exposed to zebrafish embryos and fry at a certain concentration gradient under room temperature; after the exposure, photos are taken using a high-content imaging system, and compared with a blank control group of nano-MNPs materials without any labeling and unexposed materials to evaluate the visualization performance of the fluorescent nano-EU@PS material.

[0028] The application of the double-tracer polystyrene microspheres obtained by the above-mentioned method for preparing double-tracer polystyrene microspheres containing a core-shell structure and a europium complex as nano-fluorescent EU@PS materials in biomedical research is characterized in that the nano-fluorescent EU@PS material is exposed to Chlamydomonas reinhardtii cells at a certain concentration gradient under room temperature; after the exposure, the cells are collected by centrifugation, the uptake of the material by Chlamydomonas reinhardtii is detected after acid digestion, and the material is compared with a blank control group of unexposed material to evaluate the quantitative performance of the fluorescent nano-EU@PS material.

[0029] The double-tracer polystyrene microspheres obtained by the above-mentioned method for preparing double-tracer polystyrene microspheres containing europium complexes with a core-shell structure are used as nano-fluorescent EU@PS materials in biomedical research, characterized in that the nano-fluorescent EU@PS material is exposed to Daphnia magna at a certain concentration gradient under room temperature; after the exposure, the intake of the material by Daphnia magna is detected after acid digestion, and compared with a blank control group of unexposed materials, to evaluate the quantitative performance of the fluorescent nano-EU@PS material.

[0030] The double-tracer polystyrene microspheres obtained by the above-mentioned method for preparing double-tracer polystyrene microspheres containing core-shell structure europium complexes are used as nano-fluorescent EU@PS materials in chemical stability research, characterized in that the nano-fluorescent EU@PS material is subjected to room temperature conditions and the supernatant is collected by centrifugation at different time intervals to study the effect of lanthanide complexes on its chemical stability; and the nano-EU@PS material without any labeling, EU@PS material with the same particle size and a blank control group of unexposed material are compared.

[0031] The technical effects of the present invention are as follows: the preparation method and application of a core-shell structured double-tracer polystyrene microsphere containing europium complex of the present invention are conducive to the simultaneous realization of visual and quantitative biological tracking, that is, visualization, positioning and quantification can be achieved by using the internal europium complex, thereby expanding the application scope of EU@PS materials or improving the application convenience of EU@PS materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The present invention is a graph of the morphology and particle size distribution information of double-traced polystyrene microspheres containing europium with a core-shell structure obtained by implementing a method for preparing double-traced polystyrene microspheres containing europium with a core-shell structure of the present invention. Figure 1 Included Figure 1 a to Figure 1 f. Figure 1 The observation scale of a is 500nm. Figure 1 The observation scale of b is 200nm. Figure 1 The observation scale of c is 100nm. Figure 1 a to Figure 1 c are all pictures obtained by scanning electron microscopy (SEM). The microspheres in the pictures are also called EU@PS microspheres or europium-doped polystyrene microspheres. The microspheres have regular morphology and good dispersion, indicating that EU@PS has been initially synthesized. Figure 1 d is the normal distribution curve of the hydrodynamic size of the microspheres obtained by multiple measurements by DLS and fitted by Orgin (2021). The hydrodynamic size of the microspheres is 197±5nm. Figure 1e is the polymer dispersibility index PDI obtained by multiple measurements by DLS and fitting the normal distribution curve through Orgin (2021). The PDI is 0.048±0.02. The larger the PDI, the wider the molecular weight distribution; the smaller the PDI, the more uniform the molecular weight distribution. Figure 1 f is the Zeta potential fitted normal distribution curve obtained by multiple measurements of DLS and Orgin (2021), and the Zeta potential is -29.7±2.5mV. DLS is a dynamic light scattering characterization technology (Dynamic Light Scattering). Zeta potential refers to the potential of the shear plane, also known as the electromotive potential or electromotive potential, which is an important indicator for characterizing the stability of colloidal dispersions.

[0033] Figure 2 The invention discloses a transmission electron microscope (TEM) image of double-traced polystyrene microspheres with a core-shell structure and containing europium complexes obtained by implementing a method for preparing double-traced polystyrene microspheres with a core-shell structure and containing europium complexes of the present invention. Figure 2 Included Figure 2 a Figure 2 i. Figure 2 The observation scale of a is 50nm. Figure 2 The observation scale of b is 200nm. Figure 2 The observation scale of c is 100nm. Figure 2 a to Figure 2 An unbright central area in c is surrounded by a bright peripheral area, indicating that the Eu(DMP)3phen core is covered by PS, and EU@PS with a core-shell structure is synthesized. Eu(DMP)3phen is a europium complex, DBM is dibenzoylmethyl, phen is "1,10-phenanthroline", and PS is polystyrene. Figure 2 d shows the high angle annular dark field HAADF image (HAADF, High Angle Annular Dark Field) of EU@PS microspheres. Figure 2 The smooth surface of EU@PS microspheres can be clearly seen in d. Figure 2 e is the C element distribution diagram based on energy dispersive X-ray spectroscopy (EDS) detection. Figure 2 f is the N element distribution diagram based on energy dispersive X-ray spectroscopy (EDS) detection. Figure 2 g is the element overlay map based on energy dispersive X-ray spectroscopy (EDS) detection. Figure 2 h is the O element distribution diagram based on energy dispersive X-ray spectroscopy (EDS) detection. Figure 2 i is the EU element distribution map based on energy dispersive X-ray spectroscopy (EDS) detection.

[0034] Figure 3This is the excitation spectrum, emission spectrum of EU@PS and the distribution diagram of EU@PS in different organisms. Figure 3 Included Figure 3 a to Figure 3 d. Figure 3 a is the excitation spectrum of EU@PS. Figure 3 b is the emission spectrum of EU@PS. Figure 3 c is the distribution map of EU@PS in Tetrahymena. Figure 3 d is the distribution map of EU@PS in Chlorella. DETAILED DESCRIPTION

[0035] Below is the attached figure ( Figure 1-Figure 3 ) and Examples illustrate the present invention.

[0036] Figure 1 The present invention is a graph of the morphology and particle size distribution information of double-traced polystyrene microspheres containing europium with a core-shell structure obtained by implementing a method for preparing double-traced polystyrene microspheres containing europium with a core-shell structure of the present invention. Figure 2 The invention discloses a transmission electron microscope (TEM) image of double-traced polystyrene microspheres with a core-shell structure and containing europium complexes obtained by implementing a method for preparing double-traced polystyrene microspheres with a core-shell structure and containing europium complexes of the present invention. Figure 3 This is the excitation spectrum, emission spectrum of EU@PS and the distribution of EU@PS in different organisms. Figures 1 to 3 As shown, a method for preparing a core-shell structured double-tracer polystyrene microsphere containing a europium complex comprises the following steps: step 1, synthesizing a europium complex Eu(DBM)3phen; step 2, synthesizing EU@PS microspheres containing Eu(DBM)3phen, wherein the core particles in the microspheres are Eu(DBM)3phen dispersed in a copolymer, wherein the copolymer is formed by copolymerization of styrene and methyl methacrylate, and the shell covering the core particles is polystyrene formed by polymerization of styrene.

[0037] Step 1 includes the following steps: step 1.1, preparing solution A: dissolving 2mM EuCl3·6H2O in 20ml of anhydrous ethanol; step 1.2, preparing solution B: adding 4mM 1,10-phenanthroline into 20ml of anhydrous ethanol containing 6mM dibenzoylmethane DBM; step 1.3, heating solution A and solution B to 50-70°C respectively under magnetic stirring, slowly adding solution A to solution B to react and synthesize Eu(DBM)3phen, the reaction time is 1-2h, and then cooling the reaction solution to room temperature; step 1.4, filtering the reaction solution to obtain Eu(DBM)3phen.

[0038] Step 2 includes the following steps: step 2.1, weighing 40-60g of Eu(DMP)3phen and dissolving it in 0.4-0.6ml of methyl methacrylate, and adding 1.4-1.6ml of styrene to obtain 2ml of mixed solution; step 2.2, adding 37ml of deionized water and 2ml of the mixed solution into a 250ml three-necked flask, and ultrasonicating for 10min to make the solution in the flask evenly dispersed; step 2.3, connecting the interfaces of the three-necked flask to a spherical condenser, a mechanical stirring paddle and a glass air guide tube, the glass air guide tube is connected to nitrogen, and the mechanical stirring speed is 200-300rpm; step 2.4, after passing nitrogen for 30min, heating the constant temperature oil bath pot to 70-80°C, adding 2ml of a 10mg / ml potassium persulfate aqueous solution to the three-necked flask In a bottle, the reaction generates a microsphere solution, and the reaction time is 10 hours; step 2.5, after the microsphere solution is cooled to room temperature, it is transferred to a centrifuge tube, and centrifuged at a speed of 5000-10000 rpm for 10 minutes using a high-speed centrifuge to remove the europium complex in the unreacted raw materials in the system; step 2.6, using an ultra-high-speed centrifuge at a speed of 25000-30000 rpm for 15 minutes to remove styrene and methyl methacrylate in the unreacted raw materials in the system, and the lower layer of solid material in the centrifuge tube, namely EU@PS microspheres, is collected, and the collected EU@PS microspheres are added to deionized water and dispersed by ultrasound; after repeating the centrifugation procedure three times, the EU@PS microspheres are redispersed in deionized water and stored as samples in light-proof glass bottles, and stored in a refrigerator at 4°C.

[0039] Step 1.3 includes using a magnetic stirring heater. Step 1.3 includes adjusting the pH to pH 6 to pH 7 after the A solution and the B solution are completely mixed. The mechanical stirring paddle in step 2.3 is made of polytetrafluoroethylene material.

[0040] The double-tracer polystyrene microspheres obtained by the above-mentioned method for preparing double-tracer polystyrene microspheres containing a core-shell structure europium complex are used as nano-fluorescent EU@PS materials in biomedical research, characterized in that the nano-fluorescent EU@PS material is exposed to Chlamydomonas reinhardtii cells at a certain concentration gradient under room temperature; after the exposure, the cells are collected by centrifugation, photographed using a laser confocal microscope and a high-content imaging system, and compared with a blank control group of nano-MNPs materials without any labeling and unexposed materials to evaluate the visualization performance of the fluorescent nano-EU@PS material.

[0041] The double-tracer polystyrene microspheres obtained by the above-mentioned method for preparing double-tracer polystyrene microspheres containing a core-shell structure europium complex are used as nano-fluorescent EU@PS materials in biomedical research, characterized in that the nano-fluorescent EU@PS material is exposed to Daphnia magna at a certain concentration gradient at room temperature; after the exposure, the photo is taken by a high-content imaging system-(captured), and compared with a blank control group of nano-MNPs materials without any labeling and unexposed materials to evaluate the visualization performance of the fluorescent nano-EU@PS material.

[0042] The double-tracer polystyrene microspheres obtained by the above-mentioned method for preparing double-tracer polystyrene microspheres containing core-shell structure europium complexes are used as nano-fluorescent EU@PS materials in biomedical research, characterized in that the nano-fluorescent EU@PS material is exposed to zebrafish embryos and fry at a certain concentration gradient under room temperature; after the exposure, photos are taken using a high-content imaging system, and compared with a blank control group of nano-MNPs materials without any labeling and unexposed materials to evaluate the visualization performance of the fluorescent nano-EU@PS material.

[0043] The application of the double-tracer polystyrene microspheres obtained by the above-mentioned method for preparing double-tracer polystyrene microspheres containing a core-shell structure and a europium complex as nano-fluorescent EU@PS materials in biomedical research is characterized in that the nano-fluorescent EU@PS material is exposed to Chlamydomonas reinhardtii cells at a certain concentration gradient under room temperature; after the exposure, the cells are collected by centrifugation, the uptake of the material by Chlamydomonas reinhardtii is detected after acid digestion, and the material is compared with a blank control group of unexposed material to evaluate the quantitative performance of the fluorescent nano-EU@PS material.

[0044] The double-tracer polystyrene microspheres obtained by the above-mentioned method for preparing double-tracer polystyrene microspheres containing europium complexes with a core-shell structure are used as nano-fluorescent EU@PS materials in biomedical research, characterized in that the nano-fluorescent EU@PS material is exposed to Daphnia magna at a certain concentration gradient under room temperature; after the exposure, the intake of the material by Daphnia magna is detected after acid digestion, and compared with a blank control group of unexposed materials, to evaluate the quantitative performance of the fluorescent nano-EU@PS material.

[0045] The double-tracer polystyrene microspheres obtained by the above-mentioned method for preparing double-tracer polystyrene microspheres containing core-shell structure europium complexes are used as nano-fluorescent EU@PS materials in chemical stability research, characterized in that the nano-fluorescent EU@PS material is subjected to room temperature conditions and the supernatant is collected by centrifugation at different time intervals to study the effect of lanthanide complexes on its chemical stability; and the nano-EU@PS material without any labeling, EU@PS material with the same particle size and a blank control group of unexposed material are compared.

[0046] The present invention aims at the deficiencies in the prior art, in particular, does not change the inherent MNPs properties, especially the surface properties, and provides a preparation method and application of a core-shell structured double-traced EU@PS microsphere material.

[0047] The present invention relates to the technical field of materials for synthesizing polystyrene microspheres with a core-shell structure by using a europium complex (tris(1,3-diphenyl-1,3-propanedione)mono(1,10-phenanthroline)europium(III)) as a bifunctional tracer, in particular to europium-doped polystyrene microsphere (EU@PS) materials, preparation methods and applications thereof.

[0048] according to Figure 1 Analyze its particle size morphology and particle size distribution; through different observation scales ( Figure 1 a: 500nm, b: 200nm, c: 100nm) confirmed that its morphology was regular and the dispersion was good, indicating that EU@PS was initially synthesized. Multiple measurements by DLS and fitting of the normal distribution curve by Orgin (2021) showed that the hydrodynamic size of EU@PS microspheres was 197±5nm ( Figure 1 d), polymer dispersibility index (PDI): 0.048±0.02 ( Figure 1 e); Zeta potential: -29.7±2.5mV( Figure 1 f).

[0049] Figure 1 The figure includes the morphology and particle size distribution information of EU@PS microspheres, where ac: EU@PS SEM images, d: hydrodynamic size (nm), e: PDI, and f: Zeta potential (mV).

[0050] Figure 2 Through different observation scales ( Figure 2The results of scanning transmission electron microscopy (a: 50nm, b: 200nm, c: 100nm) clearly show that a dark central area is surrounded by a bright peripheral area, indicating that the Eu(DMP)3phen core is covered by PS to synthesize EU@PS with a core-shell structure. Compared with labeling techniques such as swelling method and doping, the core-shell structure can maintain the inherent MNPs properties, especially the surface properties. In addition, the core-shell structure of EU@PS greatly avoids the leakage of europium complexes, which provides a feasible solution for clarifying the environmental behavior of MNPs in actual environments. Figure 2 d shows the high angle annular dark field (HAADF) image of EU@PS, which clearly shows the smooth surface of EU@PS. Based on energy dispersive X-ray spectroscopy (EDS), the distribution of different elements was detected (Figure (e) C, (f) N, (h) O, (i) EU, (g) element overlay), and the results showed that it was well coated inside PS.

[0051] Figure 2 Includes transmission electron microscopy observation of EU@PS microspheres, where ac: bright field, d: high-angle annular dark field (HAADF), ci: energy dispersive X-ray spectroscopy (EDS), (e) C, (f) N, (h) O, (i) EU, g: element overlay map.

[0052] Figure 3 Including the fluorescence properties and quantitative data of a nano fluorescent EU@PS microsphere. Based on the EU@PS synthesized above, its excitation / emission spectrum is further analyzed. Figure 3 aThe peak value of its excitation wavelength is 345nm, and the peak value of its emission wavelength is 613nm. It can excite 580, 592, 613, 652 and 703nm emission light under 270 and 345nm excitation light, but it is not as effective as 613nm ( Figure 2 b) has a narrow emission peak with the highest peak intensity. Therefore, the emission wavelength of EU@PS was determined to be 613nm; when using molecular fluorescence spectroscopy, 270nm is easily affected by solvents (such as water), so the excitation wavelength selected for material determination is 345nm. Using a laser confocal microscope, the distribution of EU@PS in different organisms was observed after Tetrahymena and Chlorella were exposed to a certain concentration of the above-mentioned EU@PS solution for 48 hours. The results showed that EU@PS can enter different organisms ( Figure 3 c: Tetrahymena, Figure 3 d: Chlorella).

[0053] Figure 3Includes the excitation spectrum, emission spectrum of EU@PS and the distribution of EU@PS in different organisms, where a: excitation spectrum, b: emission spectrum, c: distribution of EU@PS in Tetrahymena, d: distribution of EU@PS in Chlorella.

[0054] A double-tracing EU@PS microsphere material with a core-shell structure, including a nanoscale europium complex as a dual-quantitative metal fluorescent core, which can achieve dual quantification through inductively coupled plasma mass spectrometry (ICP-MS) and molecular fluorescence spectroscopy; the europium complex is further dispersed in the shell solution, and the shell solution-styrene is copolymerized with a certain amount of methyl methacrylate, and the unreacted styrene is once again coated with a layer of PS shell outside the core through polymerization reaction. The specific solution is:

[0055] The polymerization process includes two-step emulsion polymerization, in which the core particles are first synthesized, and then the core particles are dispersed into the shell solution to further generate the shell. Specific steps:

[0056] The first step is to synthesize the europium complex—Eu(DBM)3phen

[0057] 1. Solution A: Dissolve 2mM EuCl3·6H2O in 20ml anhydrous ethanol;

[0058] 2. Solution B: Add 4 mM 1,10-phenanthroline (Phen) to 20 ml of anhydrous ethanol containing 6 mM dibenzoylmethane (DBM);

[0059] 3. Heat the solution to 50-70°C under magnetic stirring (e.g., using a magnetic stirring heater), slowly add solution A to solution B, adjust the pH to 6-7 (e.g., using 2M NaOH solution), react for 1-2 hours; cool to room temperature;

[0060] 4. The cooled solution was filtered using a solvent filter (Jin Teng), passed through a 0.22 μm glass fiber filter membrane, rinsed three times with anhydrous ethanol, and rinsed three times with deionized water (18.2 MΩ·cm) to obtain the europium complex Eu(DBM)3phen.

[0061] The second step is to synthesize EU@PS containing europium complexes by soap-free emulsion polymerization.

[0062] 1. Weigh 40-60 g of the obtained Eu(DMP)3phen and dissolve it in 0.4-0.6 ml of methyl methacrylate, and add 1.4-1.6 ml of styrene (St) to obtain 2 ml of a mixed solution;

[0063] 2. Add 37 mL of deionized water (18.2 MΩ·cm) and 2 mL of the mixture in 1 into a 250 mL three-necked flask, and ultrasonicate for 10 min to make the solution in the flask evenly dispersed;

[0064] 3. The interfaces of the three-necked flask are connected to a spherical condenser, a mechanical stirring paddle (polytetrafluoroethylene) and a glass gas guide tube. The glass tube is connected to nitrogen. The mechanical stirring is set to 200-300 rpm.

[0065] 4. After nitrogen flow for 30 min, heat the constant temperature oil bath to 70-80 °C, add 2 mL of potassium persulfate aqueous solution (concentration 10 mg / mL), and react for 10 h;

[0066] 5. After the microsphere solution is cooled to room temperature, it is transferred to a centrifuge tube and centrifuged at 5000-10000 rpm for 10 min using a high-speed centrifuge to remove the unreacted raw material europium complex in the system;

[0067] 6. Use an ultra-high speed centrifuge at 25000-30000rpm for 15min to remove unreacted raw materials (such as styrene and methyl methacrylate) in the system, collect the EU@PS (Europium-doped polystyrene microspheres) solid material in the lower layer of the centrifuge tube, add the collected EU@PS to deionized water (18.2MΩ·cm), and further redisperse it by ultrasound; repeat the centrifugation procedure three times and redisperse it in deionized water. The sample was stored in a light-proof glass bottle and kept in a refrigerator at 4°C.

[0068] Scope of application:

[0069] An application of the above-mentioned nano fluorescent EU@PS material in biomedical research is characterized in that the nano fluorescent EU@PS material is exposed to Chlamydomonas reinhardtii cells at a certain concentration gradient under room temperature; after the exposure, the cells are collected by centrifugation, photographed by laser confocal microscopy and high content imaging system, and compared with a blank control group of nano MNPs materials without any labeling and unexposed materials to evaluate the visualization performance of the fluorescent nano EU@PS material.

[0070] An application of the above-mentioned nano fluorescent EU@PS material in biomedical research is characterized in that the nano fluorescent EU@PS material is exposed to Daphnia magna at a certain concentration gradient under room temperature; after the exposure, it is photographed by a high content imaging system-(captured), and compared with a blank control group of nano MNPs materials without any labeling and unexposed materials to evaluate the visualization performance of the fluorescent nano EU@PS material.

[0071] An application of the above-mentioned nano fluorescent EU@PS material in biomedical research is characterized in that the nano fluorescent EU@PS material is exposed to zebrafish embryos and fry at a certain concentration gradient under room temperature; after the exposure, photos are taken using a high-content imaging system, and compared with a blank control group of nano MNPs materials without any labeling and unexposed materials to evaluate the visualization performance of the fluorescent nano EU@PS material.

[0072] An application of the above-mentioned nano fluorescent EU@PS material in biomedical research is characterized in that the nano fluorescent EU@PS material is exposed to Chlamydomonas reinhardtii cells at a certain concentration gradient under room temperature; after the exposure, the cells are collected by centrifugation, the uptake of the material by Chlamydomonas reinhardtii is detected after acid digestion, and compared with a blank control group of unexposed materials to evaluate the quantitative performance of the fluorescent nano EU@PS material.

[0073] An application of the above-mentioned nano fluorescent EU@PS material in biomedical research is characterized in that the nano fluorescent EU@PS material is exposed to Daphnia magna at a certain concentration gradient under room temperature; after the exposure, the uptake of the material by Daphnia magna is detected after acid digestion, and compared with a blank control group of unexposed materials to evaluate the quantitative performance of the fluorescent nano EU@PS material.

[0074] An application of a fluorescent nano EU@PS material in a study of chemical stability, characterized in that the nano fluorescent EU@PS material is subjected to a method of collecting the supernatant by centrifugation at different time intervals at room temperature to study the effect of lanthanide complexes on its chemical stability; and the material is compared with a blank control group of unlabeled nano EU@PS material, EU@PS material of the same particle size, and unexposed material.

[0075] The contents not described in detail in the specification of the present invention belong to the prior art known to the professional and technical personnel in the field. It is pointed out here that the above description helps those skilled in the art to understand the invention, but does not limit the protection scope of the invention. Any equivalent replacement, modification and / or simplification of the above description without departing from the essence of the invention falls within the protection scope of the invention.

Claims

1. A method for preparing double-traced polystyrene microspheres containing a core-shell structured europium complex, characterized in that: The following steps are involved: Step 1, synthesizing the europium complex Eu(DBM)3phen; Step 2, synthesizing EU@PS microspheres containing Eu(DBM)3phen, wherein the core particles in the microspheres are Eu(DBM)3phen dispersed in a copolymer, wherein the copolymer is formed by copolymerization of styrene and methyl methacrylate, and the shell covering the core particles is polystyrene formed by polymerization of styrene.

2. The method for preparing double-traced polystyrene microspheres containing europium complexes with core-shell structures according to claim 1, characterized in that: Step 1 includes the following steps: Step 1.1, prepare solution A: dissolve 2 mM EuCl3·6H2O in 20 ml of anhydrous ethanol; Step 1.2, prepare solution B: add 4 mM 1,10-phenanthroline to 20 ml of anhydrous ethanol containing 6 mM dibenzoylmethane DBM; Step 1.3, heating solution A and solution B to 50-70° C. respectively under magnetic stirring, slowly adding solution A to solution B to react and synthesize Eu(DBM)3phen, the reaction time is 1-2 hours, and then cooling the reaction solution to room temperature; Step 1.4, filtering the reaction solution to obtain Eu(DBM)3phen.

3. The method for preparing double-traced polystyrene microspheres containing europium complexes with core-shell structures according to claim 1, characterized in that: Step 2 includes the following steps: Step 2.1, weigh 40-60 g of Eu(DMP)3phen and dissolve it in 0.4-0.6 ml of methyl methacrylate, and add 1.4-1.6 ml of styrene to obtain 2 ml of a mixed solution; Step 2.2, add 37 mL of deionized water and 2 mL of the mixed solution into a 250 mL three-necked flask, and ultrasonicate for 10 min to make the solution in the flask evenly dispersed; Step 2.3, the interfaces of the three-necked flask are respectively connected to a spherical condenser, a mechanical stirring paddle and a glass gas guide tube, the glass gas guide tube is connected to nitrogen, and the mechanical stirring speed is 200-300 rpm; Step 2.4, after nitrogen is passed for 30 min, the constant temperature oil bath is heated to 70-80°C, 2 mL of 10 mg / mL potassium persulfate aqueous solution is added to the three-necked flask to react to generate a microsphere solution, and the reaction time is 10 h; Step 2.5, after the microsphere solution is cooled to room temperature, it is transferred to a centrifuge tube and centrifuged at 5000-10000 rpm for 10 min using a high-speed centrifuge to remove the europium complex in the unreacted raw material in the system; In step 2.6, an ultra-high speed centrifuge is used to centrifuge at a speed of 25000-30000 rpm for 15 min to remove styrene and methyl methacrylate in the unreacted raw materials in the system, and the lower solid material in the centrifuge tube, i.e., EU@PS microspheres, is collected. The collected EU@PS microspheres are added to deionized water and dispersed by ultrasound; after repeating the centrifugation procedure three times, the EU@PS microspheres are redispersed in deionized water and stored as samples in light-proof glass bottles and stored in a refrigerator at 4°C.

4. The method for preparing double-traced polystyrene microspheres containing europium complexes with core-shell structures according to claim 2, characterized in that: Step 1.3 includes using a magnetic stirring heater; step 1.3 includes adjusting the pH to pH 6 to pH 7 after solution A and solution B are completely mixed.

5. Application of the double-tracer polystyrene microspheres obtained by the method for preparing double-tracer polystyrene microspheres containing europium complexes with a core-shell structure as claimed in any one of claims 1 to 4 as nano fluorescent EU@PS materials in biomedical research, characterized in that: The nano fluorescent EU@PS material was exposed to Chlamydomonas reinhardtii cells at a certain concentration gradient under room temperature. After the exposure, the cells were collected by centrifugation, photographed using a laser confocal microscope and a high-content imaging system, and compared with a blank control group of unlabeled nano MNPs materials and unexposed materials to evaluate the visualization performance of the fluorescent nano EU@PS material.

6. Application of the double-tracer polystyrene microspheres obtained by the method for preparing double-tracer polystyrene microspheres containing europium complexes with a core-shell structure as claimed in any one of claims 1 to 4 as nano fluorescent EU@PS materials in biomedical research, characterized in that: The nano fluorescent EU@PS material was exposed to Daphnia magna at a certain concentration gradient at room temperature. After the exposure, photos were taken using a high-content imaging system-(captured) and compared with a blank control group of nano MNPs materials without any labeling and unexposed materials to evaluate the visualization performance of the fluorescent nano EU@PS material.

7. Application of the double-tracer polystyrene microspheres obtained by the method for preparing double-tracer polystyrene microspheres containing europium complexes with a core-shell structure as claimed in any one of claims 1 to 4 as nano fluorescent EU@PS materials in biomedical research, characterized in that: The nano fluorescent EU@PS material was exposed to zebrafish embryos and larvae at a certain concentration gradient at room temperature. After the exposure, photos were taken using a high-content imaging system and compared with the blank control group of nano MNPs materials without any labeling and unexposed materials to evaluate the visualization performance of the fluorescent nano EU@PS material.

8. Application of the double-tracer polystyrene microspheres obtained by the method for preparing double-tracer polystyrene microspheres containing europium complexes with a core-shell structure as claimed in any one of claims 1 to 4 as nano fluorescent EU@PS materials in biomedical research, characterized in that: The nano-fluorescent EU@PS material was exposed to Chlamydomonas reinhardtii cells at a certain concentration gradient at room temperature. After the exposure, the cells were collected by centrifugation and the uptake of the material by Chlamydomonas reinhardtii was detected after acid digestion. The material was compared with the blank control group of unexposed material to evaluate the quantitative performance of the fluorescent nano-EU@PS material.

9. Application of the double-tracer polystyrene microspheres obtained by the method for preparing double-tracer polystyrene microspheres containing europium complexes with a core-shell structure as claimed in any one of claims 1 to 4 as nano fluorescent EU@PS materials in biomedical research, characterized in that: The nano fluorescent EU@PS material was exposed to Daphnia magna at a certain concentration gradient under room temperature. After the exposure, the intake of the material by Daphnia magna was detected by acid digestion and compared with the blank control group of unexposed material to evaluate the quantitative performance of the fluorescent nano EU@PS material.

10. The double-tracer polystyrene microspheres obtained by the method for preparing double-tracer polystyrene microspheres containing europium complexes with core-shell structures as claimed in any one of claims 1 to 4 are used as nano fluorescent EU@PS materials in the study of chemical stability, characterized in that: The nano-fluorescent EU@PS material was centrifuged at room temperature and the supernatant was collected at different time intervals to study the effect of lanthanide complexes on its chemical stability; and the material was compared with the blank control group of unlabeled nano-EU@PS material, EU@PS material of the same particle size and unexposed material.

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