Rare earth upconversion red-green-blue fluorescent encoding microspheres and preparation method thereof

Abstract

The application discloses a rare earth up-conversion red-green-blue fluorescent coding microsphere, adopts a modified olefin-norbornene copolymer as a matrix material, and adopts red, blue or green rare earth up-conversion luminescent nanocrystals as a fluorescent coding material. The application further discloses a preparation method of the rare earth up-conversion red-green-blue fluorescent coding microsphere. The modified olefin-norbornene copolymer is blended with the up-conversion luminescent nanocrystals to obtain a uniform blend, and the blend is hot-pressed into a rod-shaped core layer which is inserted into a cladding material to form a preform rod, and then the preform rod is hot-drawn into a fiber, and then heat treatment is carried out to obtain a polymer fluorescent microsphere with a coding function. The method can control the fluorescent type and intensity of the fluorescent coding microsphere and the particle size of the microsphere according to requirements, the preparation process is simple, the method is suitable for large-scale production, the fluorescent performance is stable, and the method can be used for detecting cells, proteins or other molecules in biological samples.

Description

Technical Field

[0001] This invention relates to the field of fluorescently encoded microspheres, specifically to a rare-earth upconversion red-green-blue fluorescently encoded microsphere and its preparation method. Background Technology

[0002] Fluorescently encoded microsphere detection technology is one of the most promising multi-index detection technologies in recent years. Fluorescently encoded microspheres can simultaneously identify and quantify nucleic acids, proteins, and small molecule compounds. Furthermore, this technology offers numerous advantages, such as highly controllable microsphere size and fluorescence intensity, high sensitivity and specificity, good biocompatibility, simple encoding and decoding, quantitative detection, high fluorescence stability, and ease of preparation and operation. Therefore, fluorescently encoded microsphere detection technology has broad application prospects in biomedicine, biopharmaceuticals, cancer diagnosis, drug release and targeted detection, monitoring of hazardous substances in the environment, and environmental pollution control.

[0003] Compared to traditional organic fluorescent dyes and inorganic quantum dots, rare-earth upconversion luminescent nanocrystals exhibit longer luminescence lifetimes, are less susceptible to decay due to environmental factors, possess better luminescence stability, are less prone to photobleaching from light radiation, and typically have narrower emission spectra, providing a purer spectrum. These characteristics make them more suitable for preparing fluorescently encoded microspheres. Polymer fluorescent microspheres loaded with rare-earth upconversion luminescent nanocrystals are suitable for long-term tracking and labeling applications, and are particularly suitable for long-term stable fluorescent labeling, exhibiting excellent fluorescence stability under various environmental conditions.

[0004] Currently, the main methods for preparing fluorescently encoded microspheres include swelling, embedding, and covalent bonding. Fluorescently encoded microspheres prepared by swelling are prone to leakage of nanocrystals from the microsphere interior. Embedding inevitably introduces free radicals, which can cause defects and significant fluorescence quenching. Covalent bonding involves a cumbersome experimental process, and nanocrystals are prone to non-specific adsorption on the microsphere surface.

[0005] Furthermore, research has found that olefin-norbornene copolymers are suitable as matrix materials for fluorescently encoded microspheres due to their advantages such as easy processing, low manufacturing cost, easy modification of channel surfaces, and good biocompatibility, as well as their good chemical inertness, good optical transparency, and low fluorescence background. However, the inner walls of the channels in olefin-norbornene copolymers are highly hydrophobic, while rare-earth upconversion nanocrystals have ligands on their surfaces. This makes phase separation easy to occur when the two are mixed, causing the nanocrystals to aggregate in the molten copolymer. This results in poor monodispersity of the fluorescently encoded microspheres and a tendency for them to stick together, which significantly reduces the sensitivity of the fluorescently encoded microspheres. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention discloses a rare-earth upconversion red-green-blue fluorescent encoded microsphere. The microsphere uses a modified olefin-norbornene copolymer as the matrix material and red, blue, or green rare-earth upconversion luminescent nanocrystals as the fluorescent encoding material. Furthermore, this invention also discloses a method for preparing rare-earth upconversion red-green-blue fluorescent encoded microspheres. This involves modifying the olefin-norbornene copolymer, blending the modified olefin-norbornene copolymer with upconversion luminescent nanocrystals to obtain a uniform blend, hot-pressing it into a rod shape as the core layer, inserting it into a primary cladding rod to form a primary preform, and then hot-drawing it into a primary fiber. Multiple fibers are then inserted into a secondary cladding rod to form a secondary preform, which is then hot-drawn into a secondary fiber. The secondary fibers, after secondary stretching, are heat-treated to obtain polymer fluorescent microspheres with encoding function. During preparation, the type and concentration of the rare-earth upconversion luminescent nanocrystals can be adjusted to allow the encoded microspheres to exhibit different types and intensities of fluorescence, resulting in monochromatic and multicolor fluorescent encoded microspheres with ultra-large encoding capacity. The method of this invention has a simple process flow, is suitable for large-scale production, has a large microsphere encoding capacity, and stable fluorescence performance, and can be used to detect cells, proteins or other molecules in biological samples.

[0007] This invention discloses a rare-earth upconversion red-green-blue fluorescent encoded microsphere, comprising the following components:

[0008] Z1wt% modified olefin-norbornene copolymer, 75≤Z1<100;

[0009] Z2wt% red upconversion luminescent nanocrystals, 0≤Z2≤25;

[0010] Z3wt% green upconversion luminescent nanocrystals, 0≤Z3≤25;

[0011] Z4wt% blue upconversion luminescent nanocrystals, 0≤Z4≤25; Z2, Z3, and Z4 are not simultaneously 0;

[0012] The modified olefin-norbornene copolymer has the following structural formula:

[0013] 1≤X≤10, 1≤Y≤10, R is a hydroxyl or amino group.

[0014] A further technical solution is: the structural formula of the red upconversion luminescent nanocrystals is CuSc. 0.81 In 0.99 F8:0.19Er 3+ 0.01Mg 2+ The structural formula of the green upconversion luminescent nanocrystals is K3Sr. 3.76 In5F 260.2Yb, 0.02Er, 0.02Pr; The structural formula of the blue upconversion luminescent nanocrystal is Ni8Sc. 2.45 Al 2.55 F8:0.55Tm 3+ 0.45Mg 2+ .

[0015] This invention also discloses a method for preparing rare-earth upconversion red-green-blue fluorescent encoded microspheres, comprising the following steps:

[0016] Step 1: Add the olefin-norbornene copolymer to a toluene solution, heat and stir at 50°C for 25-40 minutes until completely dissolved to obtain a mixed solution, and then use a coating machine to form a film from the mixed solution;

[0017] Step 2: Crush the film from Step 1 and add it to bromine water. React under ultraviolet light for 12 hours, then soak it in an alkaline solution for 24 hours. Freeze-dry the reaction solution at -99℃ to obtain the modified olefin-norbornene copolymer.

[0018] Step 3: Weigh Z1wt% of the modified olefin-norbornene copolymer from Step 2 and add it to a beaker containing cyclohexane solvent. Heat and stir continuously at 60℃. Weigh Z2wt% of red upconversion nanocrystals, Z3wt% of green upconversion luminescent nanocrystals, and Z4wt% of blue upconversion luminescent nanocrystals and disperse them in a container containing cyclohexane solvent. After the modified olefin-norbornene copolymer is completely dissolved, pour the mixed solution from the container into the beaker and stir continuously for 8–12 h. Then, sonicate in an ice-water bath for 2–3 h, and freeze-dry at -30 to -10℃ for 15–24 h to obtain the core material. Wherein, 75≤Z1<100, 0≤Z2≤25, 0≤Z3≤25, 0≤Z4≤25; Z2, Z3, and Z4 are not simultaneously 0.

[0019] Step 4: Hot-press the cladding material into a cylindrical primary cladding rod with a cavity. Hot-press the core material from Step 3 into a cylindrical core rod at 230°C. Place the core rod into the cavity of the primary cladding rod, and then place the primary cladding rod in a vacuum drying oven for heat curing at 100°C for 30 minutes to obtain a primary preform. Hot-draw the primary preform at 360°C to obtain primary fibers.

[0020] Step 5: Hot-press the cladding material into a cylindrical secondary cladding rod with an internal cavity. Take multiple primary fibers with the same diameter and height and insert them into the cavity of the secondary cladding rod. Then place the secondary cladding rod in a vacuum drying oven and heat-cur it at 100°C for 30 minutes to obtain a secondary preform. Heat-draw the secondary preform at 360°C to obtain secondary fibers.

[0021] Step 6: Heat-treat the secondary fibers from Step 5 at 350–380℃ for 3–10 minutes;

[0022] Step 7: Dissolve the secondary fibers from Step 6 in the selective solvent for the coating material, heat and stir until completely dissolved, centrifuge at high speed for 5 minutes, remove the supernatant and collect the precipitate, add the precipitate back into the selective solvent for the coating material, and after repeated dissolution, centrifugation and filtration, the coating material is completely removed to obtain rare earth upconversion red-green-blue fluorescent encoded microspheres.

[0023] A further technical solution is: the structural formula of the red upconversion luminescent nanocrystal is as follows:

[0024] CuSc 0.81 In 0.99 F8:0.19Er 3+ 0.01Mg 2+ The structural formula of the green upconversion luminescent nanocrystals is:

[0025] K3Sr 3.76 In5F 26 The structural formula of the blue upconversion luminescent nanocrystals is: 0.2Yb, 0.02Er, 0.02P.

[0026] Ni8Sc 2.45 Al 2.55 F8:0.55Tm 3+ 0.45Mg 2+ .

[0027] A further technical solution is that the cladding material is polysulfone resin.

[0028] A further technical solution is that the selective solvent for the cladding material is one of N,N-dimethylacetamide, N-methylpyrrolidone, N,N-dimethylformamide, or chloroform.

[0029] A further technical solution is that the alkaline solution is one of potassium hydroxide, sodium hydroxide, or ethylenediamine solution.

[0030] A further technical solution is that the thickness of the film obtained in step 1 is 200–400 μm.

[0031] A further technical solution is that the diameter of the primary cladding rod in step 4 and the secondary cladding rod in step 5 is 20-30 mm, and the diameter of the core rod in step 4 is 2-4 mm.

[0032] A further technical solution is that the diameter of the primary fiber in step 4 and the secondary fiber in step 5 is 400-800 μm.

[0033] A further technical solution is that the microspheres obtained in step 7 have a particle size of 5–10 μm.

[0034] The beneficial effects of this invention are:

[0035] This invention modifies the olefin-norbornene copolymer by nucleophilic substitution to attach -OH or -NH2 functional groups to its surface. The modified olefin-norbornene copolymer exhibits a significantly reduced surface contact angle, effectively mitigating its strong hydrophobicity and thus improving the dispersibility of rare-earth upconversion luminescent nanocrystals, thereby enhancing the performance of fluorescently encoded microspheres. Furthermore, this invention utilizes a solution blending method to mix the modified olefin-norbornene copolymer and rare-earth upconversion luminescent nanocrystals, further promoting the dispersibility of the rare-earth upconversion luminescent nanocrystals within the modified olefin-norbornene copolymer. Furthermore, this invention employs a fiber core-layer hydrodynamic preparation method. Utilizing the instability of the fluid within the fiber, the glass transition temperature difference between the cladding and core materials, the viscosity of the core material, and the selective dissolution of the cladding material by the solvent, the core layer, composed of a modified olefin-norbornene copolymer and a blend of rare-earth upconversion luminescent nanocrystals, softens and shrinks into polymer fluorescent microspheres. The microspheres prepared by this method exhibit good monodispersity, uniform particle size, and a wide controllable size range, effectively solving the problem of adhesion in fluorescently encoded microspheres. The microsphere preparation process of this invention involves only physical processes and not chemical processes; therefore, the obtained fluorescently encoded microspheres highly retain the physicochemical properties of the rare-earth upconversion luminescent nanocrystals.

[0036] In the preparation of the microspheres of this invention, different types and concentrations of nanocrystals can be used as the encoding base material, resulting in microspheres with different types and intensities of fluorescence. This leads to monochromatic and multicolor fluorescent encoded microspheres with ultra-high encoding capacity, enabling fluorescence encoding and high-throughput detection. Furthermore, the particle size of the prepared microspheres can be adjusted by controlling the number of fiber stretching cycles, thereby meeting the needs of various experiments. Attached Figure Description

[0037] Figure 1 The particle size distribution and scanning electron microscope image of the pure red fluorescent coded microspheres in Example 1;

[0038] Figure 2 The fluorescence spectrum of the pure red fluorescently encoded microspheres in Example 1;

[0039] Figure 3 The particle size distribution and scanning electron microscope image of the pure green fluorescent coded microspheres in Example 2;

[0040] Figure 4 The fluorescence spectrum of the pure green fluorescent encoded microspheres in Example 2;

[0041] Figure 5The particle size distribution and scanning electron microscope image of the pure blue fluorescently encoded microspheres in Example 3;

[0042] Figure 6 The fluorescence spectrum of the pure blue fluorescently encoded microspheres in Example 3;

[0043] Figure 7 The upconversion fluorescence spectrum of the red and green dual-color fluorescent coded microspheres in Example 4 is shown below.

[0044] Figure 8 The upconversion fluorescence spectrum of the red and blue dual-color fluorescent coded microspheres in Example 5 is shown below.

[0045] Figure 9 The upconversion fluorescence spectrum of the green and blue dual-color fluorescent coded microspheres in Example 6 is shown below.

[0046] Figure 10 The image shows the morphology and particle size distribution histogram of the red, green, and blue multicolor fluorescent coded microspheres in Example 7 under an optical microscope.

[0047] Figure 11 The upconversion fluorescence spectrum of the red, green, and blue multicolor fluorescent coded microspheres in Example 7 is shown below.

[0048] Figure 12 This is a schematic diagram of the three-dimensional matrix coding constructed from the multicolor fluorescent coded microspheres prepared in Examples 1-7. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0050] Example 1

[0051] (1) Weigh 1g of olefin-norbornene copolymer and pour it into a beaker containing 20mL of toluene solution. Stir and heat continuously at 50℃ for 30min to completely dissolve the olefin-norbornene copolymer and obtain a mixed solution. Use a coating machine to make a film with a thickness of about 400μm from the obtained mixed solution.

[0052] (2) The obtained film was cut into small pieces and poured into 3% bromine water. The mixture was reacted under UV light for 12 hours to bromine-modify the olefin-norbornene copolymer. The brominated olefin-norbornene copolymer was then immersed in 1M potassium hydroxide solution for 24 hours. A nucleophilic substitution reaction was used to attach -OH functional groups to the surface of the olefin-norbornene copolymer. The resulting solution was then freeze-dried at -99°C for 24 hours to obtain the modified olefin-norbornene copolymer, with the following structural formula: Where X = Y = 5, and R is a hydroxyl group.

[0053] The relevant properties of the obtained product were characterized. The surface contact angle of the unmodified olefin-norbornene copolymer was about 92°, while that of the modified olefin-norbornene copolymer was 45°. The results showed that the hydrophobicity of the modified olefin-norbornene copolymer was significantly reduced.

[0054] (3) Take 1g of the modified olefin-norbornene copolymer obtained in step (2) and put it into a beaker containing 20mL of cyclohexane. Heat at 60℃ and stir continuously for 60min to fully dissolve the modified olefin-norbornene copolymer. Then take 0.01g of the structure with the formula CuSc 0.81 In 0.99 F8:0.19Er 3+ 0.01Mg 2+ Pure red upconversion luminescent nanocrystals were dispersed in a container containing 1 mL of cyclohexane solvent. After the modified olefin-norbornene copolymer was completely dissolved, the solution in the container was poured into a beaker and stirred continuously for 12 h. Then, the mixture was sonicated in an ice-water bath for 2 h to uniformly disperse the pure red upconversion luminescent nanocrystals in the mixed solution containing the modified olefin-norbornene copolymer. Finally, the mixture was freeze-dried at -15 °C for 22 h to completely remove the cyclohexane. The product was collected to obtain the core material as a precursor for red fluorescently encoded microspheres. The proportion of pure red upconversion luminescent nanocrystals was approximately 1 wt%.

[0055] (4) Using a cylindrical mold, polysulfone resin is hot-pressed at 300°C to form a cylindrical primary cladding rod with a cavity. The diameter of the primary cladding rod is 20 mm, and the diameter of the cavity inside the primary cladding rod is 2 mm. Using a cylindrical mold, the core material obtained in step (3) is hot-pressed at 230°C to form a core rod with a diameter of 2 mm. The core rod is inserted into the cavity inside the primary cladding rod as the fiber core. The primary cladding rod is then placed in a vacuum drying oven and heat-cured at 100°C for 30 min to obtain a primary preform. The primary preform is placed on a drawing tower and hot-drawn at 360°C into a primary fiber with a diameter of 600 μm.

[0056] (5) The polysulfone resin material is hot-pressed at 310°C to form a secondary cladding rod with a diameter of 30 mm. A cavity with a diameter of 6 mm is set inside the secondary cladding rod. 70 fibers with the same diameter and height after heat treatment in step (5) are selected and inserted into the cavity of the secondary cladding rod. The secondary cladding rod is then placed in a vacuum drying oven and heat-cured at 100°C for 30 min to obtain a secondary preform. The secondary preform is placed on a drawing tower and hot-drawn at 360°C into a secondary fiber with a diameter of 600 μm.

[0057] (6) The secondary fiber from step (5) is placed in a tube furnace and heat-treated at 380°C for 8 minutes, so that the core of the secondary fiber reaches the glass transition temperature to form a fluid and shrinks into microspheres under the action of elasticity.

[0058] (7) The secondary fibers from step (6) were placed in a beaker containing 50 mL of N,N-dimethylacetamide solvent and stirred continuously at 80 °C. After the polysulfone resin coating material was fully dissolved, the mixture was centrifuged at 13,000 rpm for 5 min. The supernatant was extracted and the precipitate was collected. The precipitate was added back to the N,N-dimethylacetamide solvent, and the above dissolution, centrifugation, and filtration operations were repeated 8 times to fully remove the polysulfone resin coating material, finally obtaining pure red fluorescent coded microspheres with a particle size of 7 μm. As can be seen from Figure 1, the pure red fluorescent coded microspheres have a good morphology and structure, excellent monodispersity and particle size uniformity, and the microspheres are regular, smooth, and non-adhesive.

[0059] Example 2

[0060] (1) Weigh 1g of olefin-norbornene copolymer and pour it into a beaker containing 30mL of toluene solution. Stir and heat continuously at 50℃ for 25min to completely dissolve the olefin-norbornene copolymer and obtain a mixed solution. Use a coating machine to make a film with a thickness of about 200μm from the obtained mixed solution.

[0061] (2) The obtained film was cut into small pieces and poured into 3% bromine water. The mixture was reacted under UV light for 12 hours to bromine-modify the olefin-norbornene copolymer. The brominated olefin-norbornene copolymer was then immersed in 1M potassium hydroxide solution for 24 hours. A nucleophilic substitution reaction was used to attach -OH functional groups to the surface of the olefin-norbornene copolymer. The resulting solution was then freeze-dried at -99°C for 24 hours to obtain the modified olefin-norbornene copolymer, with the following structural formula: Where X = Y = 5, and R is a hydroxyl group.

[0062] The relevant properties of the obtained product were characterized. The surface contact angle of the unmodified olefin-norbornene copolymer was about 92°, while that of the modified olefin-norbornene copolymer was 42°. The results show that the hydrophobicity of the modified olefin-norbornene copolymer was significantly reduced.

[0063] (3) Take 1g of the modified olefin-norbornene copolymer obtained in step (2) and put it into a beaker containing 40mL of cyclohexane. Heat at 60℃ and stir continuously for 20min to fully dissolve the modified olefin-norbornene copolymer. Then take 0.005g of the K3Sr... 3.76 In5F 26 Pure green upconversion luminescent nanocrystals (0.2Yb, 0.02Er, 0.02Pr) were dispersed in a container containing 1 mL of cyclohexane solvent. After the modified olefin-norbornene copolymer was completely dissolved, the solution in the container was poured into a beaker and stirred continuously for 12 h. Then, the mixture was sonicated in an ice-water bath for 2 h to uniformly disperse the pure green upconversion luminescent nanocrystals in the mixed solution containing the modified olefin-norbornene copolymer. Finally, the mixture was freeze-dried at -20 °C for 24 h to completely remove the cyclohexane. The product was collected to obtain the core material, which served as a precursor for green fluorescent encoded microspheres. The proportion of pure green upconversion luminescent nanocrystals was approximately 0.5 wt%.

[0064] (4) Using a cylindrical mold, polysulfone resin is hot-pressed at 290°C to form a cylindrical primary cladding rod with a cavity. The diameter of the primary cladding rod is 20 mm, and the diameter of the cavity inside the primary cladding rod is 2 mm. The cavity is set along the length of the primary cladding rod. Using a cylindrical mold, the core material obtained in step (3) is hot-pressed at 230°C to form a core rod with a diameter of 2 mm. After inserting the core rod as the fiber core into the cavity of the primary cladding rod, the primary cladding rod is placed in a vacuum drying oven and heat-cured at 100°C for 30 min to obtain a primary preform. The primary preform obtained in step (4) is placed on a drawing tower and hot-drawn at 360°C to form a primary fiber with a diameter of 400 μm.

[0065] (5) The polysulfone resin material is hot-pressed at 300°C to form a secondary cladding rod with a diameter of 20 mm. A cavity with a diameter of 6 mm is set inside the secondary cladding rod. 70 primary fibers with the same diameter and height from step (4) are selected and inserted into the cavity of the secondary cladding rod. The secondary cladding rod is then placed in a vacuum drying oven and heat-cured at 100°C for 30 min to obtain a secondary preform. The secondary preform is then placed on a drawing tower and hot-drawn at 360°C into a secondary fiber with a diameter of 400 μm.

[0066] (6) The secondary fibers from step (5) are placed in a tube furnace and heat-treated at 280°C for 5 minutes, so that the core of the secondary fibers reaches the glass transition temperature to form a fluid and shrinks into microspheres under the action of elasticity.

[0067] (7) Place the secondary fibers from step (6) into a beaker containing 50 mL of N,N-dimethylacetamide solvent. Stir continuously at 80°C until the polysulfone resin coating material is fully dissolved. Centrifuge the mixture at 13000 rpm for 5 minutes, extract the supernatant, and collect the precipitate. Add the precipitate back into the N,N-dimethylacetamide solvent and repeat the above dissolution, centrifugation, and filtration process 7 times to thoroughly remove the polysulfone resin coating material. Figure 3 and Figure 4 As shown, pure green fluorescent encoded microspheres with a particle size of 6 μm were finally obtained.

[0068] As can be observed from Figure 3, the green fluorescent encoded microspheres have a good morphology and structure, excellent monodispersity and particle size uniformity, and a smooth surface.

[0069] Example 3

[0070] (1) Weigh 1g of olefin-norbornene copolymer and pour it into a beaker containing 40mL of toluene solution. Stir and heat continuously at 50℃ for 30min to completely dissolve the olefin-norbornene copolymer and obtain a mixed solution. Use a coating machine to make a film with a thickness of about 300μm from the obtained mixed solution.

[0071] (2) The obtained film was cut into small pieces and poured into a 3% bromine solution. The mixture was reacted under UV light for 12 hours to bromine-modify the olefin-norbornene copolymer. The brominated olefin-norbornene copolymer was then immersed in a 10% ethylenediamine aqueous solution for 24 hours. A substitution reaction was performed to attach -NH2 functional groups to the surface of the olefin-norbornene copolymer. The resulting solution was then freeze-dried at -99°C for 24 hours to obtain the modified olefin-norbornene copolymer, with the following structural formula: Where X = Y = 5, and R is an amino group.

[0072] The relevant properties of the obtained product were characterized. The surface contact angle of the unmodified olefin-norbornene copolymer was about 92°, while that of the modified olefin-norbornene copolymer was 41°. The results show that the hydrophobicity of the modified olefin-norbornene copolymer was significantly reduced.

[0073] (3) Take 1g of the modified olefin-norbornene copolymer obtained in step (2) and put it into a beaker containing 30mL of cyclohexane. Heat at 60℃ and stir continuously for 50min to fully dissolve the modified olefin-norbornene copolymer. Then take 0.15g of the structure with the formula Ni8Sc 2.45 Al 2.55 F8:0.55Tm 3+ 0.45Mg 2+Pure blue upconversion luminescent nanocrystals were dispersed in a container containing 1 mL of cyclohexane solvent. After the modified olefin-norbornene copolymer was completely dissolved, the solution in the container was poured into a beaker and stirred continuously for 12 h. Then, the mixture was sonicated in an ice-water bath for 2 h to uniformly disperse the pure blue upconversion luminescent nanocrystals in the mixed solution containing the modified olefin-norbornene copolymer. Finally, the mixture was freeze-dried at -30 °C for 24 h to completely remove the cyclohexane. The product was collected to obtain the core material as a precursor for blue fluorescently encoded microspheres. The proportion of pure blue upconversion luminescent nanocrystals was approximately 13 wt%.

[0074] (4) Using a cylindrical mold, polysulfone resin is hot-pressed at 300°C to form a cylindrical primary cladding rod with a cavity. The diameter of the primary cladding rod is 30 mm, and the diameter of the cavity inside the primary cladding rod is 2 mm. Using a cylindrical mold, the core material obtained in step (3) is hot-pressed at 230°C to form a core rod with a diameter of 2 mm. The core rod is inserted into the cavity inside the primary cladding rod as the fiber core. The primary cladding rod is then placed in a vacuum drying oven and heat-cured at 100°C for 30 min to obtain a primary preform. The primary preform is placed on a drawing tower and hot-drawn at 360°C into a primary fiber with a diameter of 500 μm.

[0075] (5) The polysulfone resin material is hot-pressed at 310°C to form a secondary cladding rod with a diameter of 30 mm. A cavity with a diameter of 6 mm is set inside the secondary cladding rod. 80 primary fibers with the same diameter and height from step (4) are selected and inserted into the cavity of the secondary cladding rod. The secondary cladding rod is then placed in a vacuum drying oven and heat-cured at 100°C for 30 min to obtain a secondary preform. The secondary preform is then placed on a drawing tower and hot-drawn at 360°C into a secondary fiber with a diameter of 500 μm.

[0076] (6) The secondary fiber is placed in a tube furnace and heat-treated at 380°C for 8 minutes, so that the core of the secondary fiber reaches the glass transition temperature to form a fluid and shrinks into microspheres under the action of elasticity.

[0077] (7) Place the secondary fibers from step (6) into a beaker containing 50 mL of N,N-dimethylacetamide solvent and stir continuously at 80 °C. After the polysulfone resin coating material is fully dissolved, centrifuge the mixture at 13,000 rpm for 5 min, extract the supernatant and collect the precipitate. Add the precipitate back into the N,N-dimethylacetamide solvent and repeat the above dissolution, centrifugation and filtration operations 8 times to fully remove the polysulfone resin coating material, finally obtaining pure blue fluorescently coded microspheres with a particle size of 5 μm. As can be observed from Figure 5, the pure blue fluorescently coded microspheres have a good morphological structure and excellent monodispersity and particle size uniformity. The microspheres are regular and smooth in shape and do not adhere to each other.

[0078] Example 4

[0079] Steps 1 and 2 in Example 4 are exactly the same as steps 1 and 2 in Example 3. The modification process and component ratio of the olefin-norbornene copolymer are the same, and will not be repeated here.

[0080] (3) Take 1g of the modified olefin-norbornene copolymer obtained in step (2) and place it in a beaker containing 35mL of cyclohexane. Heat at 60℃ and stir continuously for 35min to fully dissolve the modified olefin-norbornene copolymer. Then take 0.1g of the structure with the formula CuSc 0.81 In 0.99 F8:0.19Er 3+ 0.01Mg 2+ Pure red upconversion luminescent nanocrystals and 0.1g of the structural formula K3Sr 3.76 In5F 26 Pure green upconversion luminescent nanocrystals of 0.2Yb, 0.02Er, and 0.02Pr were dispersed in a container containing 1 mL of cyclohexane solvent. After the modified olefin-norbornene copolymer was completely dissolved, the solution in the container was poured into a beaker and stirred continuously for 12 h. Then, the mixture was sonicated in an ice-water bath for 2 h to ensure that the nanocrystals were uniformly dispersed in the mixed solution containing the modified olefin-norbornene copolymer. Finally, the mixture was freeze-dried at -20 °C for 24 h to completely remove the cyclohexane. The product was collected to obtain the core material, which served as a precursor for red and green dual-color fluorescent encoded microspheres. The proportion of red and green upconversion luminescent nanocrystals was approximately 16.7 wt%.

[0081] (4) Using a cylindrical mold, polysulfone resin is hot-pressed at 500°C to form a cylindrical primary cladding rod with a cavity. The diameter of the primary cladding rod is 30 mm, and the diameter of the cavity inside the primary cladding rod is 2 mm. The cavity is set along the length of the cladding rod. Using a cylindrical mold, the core material obtained in step (3) is hot-pressed at 230°C to form a core rod with a diameter of 2 mm. After inserting the core rod as the fiber core into the cavity of the primary cladding rod, the primary cladding rod is placed in a vacuum drying oven and heat-cured at 100°C for 30 min to obtain a primary preform. The primary preform is placed on a drawing tower and hot-drawn at 360°C into a primary fiber with a diameter of 600 μm.

[0082] (5) The polysulfone resin cladding material is hot-pressed at 300°C to form a secondary cladding rod with a diameter of 20 mm. A cavity with a diameter of 6 mm is set inside the secondary cladding rod. 70 primary fibers with the same diameter and height from step (4) are selected and inserted into the cavity of the secondary cladding rod. The secondary cladding rod is then placed in a vacuum drying oven and heat-cured at 100°C for 30 min to obtain a secondary preform. The secondary preform is then placed on a drawing tower and hot-drawn at 360°C into a secondary fiber with a diameter of 600 μm.

[0083] (6) The secondary fiber is placed in a tube furnace and heat-treated at 380°C for 10 minutes to make the core of the secondary fiber reach the glass transition temperature to form a fluid and shrink into microspheres under the action of elasticity.

[0084] (7) Place the secondary fibers from step (6) into a beaker containing 50 mL of N,N-dimethylacetamide solvent. Stir continuously at 80°C until the polysulfone resin coating material is fully dissolved. Centrifuge the mixture at 13000 rpm for 5 minutes, extract the supernatant, and collect the precipitate. Add the precipitate back into the N,N-dimethylacetamide solvent and repeat the above dissolution, centrifugation, and filtration process 7 times to thoroughly remove the polysulfone resin coating material. Figure 7 As shown, red and green bicolor fluorescent coded microspheres with a particle size of 10 μm were finally obtained.

[0085] Example 5

[0086] Steps 1 and 2 in Example 5 are exactly the same as steps 1 and 2 in Example 3. The modification process and component ratio of the olefin-norbornene copolymer are the same, and will not be repeated here.

[0087] (3) Take 1g of the modified olefin-norbornene copolymer obtained in step (2) and place it in a beaker containing 20mL of cyclohexane. Stir continuously at 60℃ for 55min to fully dissolve the modified olefin-norbornene copolymer. Then take 0.1g of the material with the structural formula CuSc... 0.81 In 0.99 F8:0.19Er 3+ 0.01Mg 2+ Pure red upconversion luminescent nanocrystals and 0.15g of Ni8Sc 2.45 Al 2.55 F8:0.55Tm 3+ 0.45Mg 2+ Pure blue upconversion luminescent nanocrystals were dispersed in a container containing 1 mL of cyclohexane solvent. After the modified olefin-norbornene copolymer was completely dissolved, the solution in the container was poured into a beaker and stirred continuously for 12 h. Then, the mixture was sonicated in an ice-water bath for 2 h to uniformly disperse the nanocrystals in the mixed solution containing the modified olefin-norbornene copolymer. Finally, the mixture was freeze-dried at -10 °C for 24 h to completely remove the cyclohexane. The product was collected to obtain the core material as a precursor for red and blue dual-color fluorescent encoded microspheres. The proportion of red and blue dual-color upconversion luminescent nanocrystals was 20 wt%.

[0088] (4) Using a cylindrical mold, polysulfone resin is hot-pressed at 500°C to form a cylindrical primary cladding rod with a cavity. The diameter of the primary cladding rod is 25 mm, and the diameter of the cavity inside the primary cladding rod is 2 mm. The cavity is set along the length of the primary cladding rod. Using a cylindrical mold, the core material obtained in step (3) is hot-pressed at 230°C to form a core rod with a diameter of 2 mm. After inserting the core rod as the fiber core into the cavity of the primary cladding rod, the primary cladding rod is placed in a vacuum drying oven and heat-cured at 100°C for 30 min to obtain a primary preform. The primary preform is placed on a drawing tower and hot-drawn at 360°C into a primary fiber with a diameter of 600 μm.

[0089] (5) A secondary cladding rod with a diameter of 20 mm is made by hot pressing polysulfone resin cladding material at 300℃. A cavity with a diameter of 6 mm is set inside the secondary cladding rod. 70 primary fibers with the same diameter and height from step (4) are selected and inserted into the cavity of the secondary cladding rod. The secondary cladding rod is then placed in a vacuum drying oven and heat-cured at 100℃ for 30 min to obtain a secondary preform. The secondary preform is then placed on a drawing tower and hot-drawn at 360℃ into a secondary fiber with a diameter of 600 μm.

[0090] (6) The secondary fibers from step (5) are placed in a tube furnace and heat-treated at 380°C for 10 minutes to allow the core of the secondary fibers to reach the glass transition temperature, form a fluid, and shrink into microspheres under the action of elasticity.

[0091] (7) Place the secondary fibers from step (6) into a beaker containing 50 mL of N,N-dimethylacetamide solvent. Stir continuously at 80°C until the polysulfone resin coating material is fully dissolved. Centrifuge the mixture at 13000 rpm for 5 minutes, extract the supernatant, and collect the precipitate. Add the precipitate back into the N,N-dimethylacetamide solvent and repeat the above dissolution, centrifugation, and filtration process 6 times to thoroughly remove the polysulfone resin coating material. Figure 8 As shown, red and blue bicolor fluorescent coded microspheres with a particle size of 10 μm were finally obtained.

[0092] Example 6

[0093] Steps 1 and 2 in Example 6 are exactly the same as steps 1 and 2 in Example 3. The modification process and component ratio of the olefin-norbornene copolymer are the same, and will not be repeated here.

[0094] (3) Take 1g of the modified olefin-norbornene copolymer obtained in step (2) and put it into a beaker containing 40mL of cyclohexane. Heat at 60℃ and stir continuously for 30min to fully dissolve the modified olefin-norbornene copolymer. Then take 0.05g of the K3Sr... 3.76 In5F 260.2Yb, 0.02Er, 0.02Pr pure green upconversion luminescent nanocrystals and 0.15g Ni8Sc 2.45 Al 2.55 F8:0.55Tm 3+ 0.45Mg 2+ Pure blue upconversion luminescent nanocrystals were dispersed in a container containing 1 mL of cyclohexane solvent. After the modified olefin-norbornene copolymer was completely dissolved, the solution in the container was poured into a beaker and stirred continuously for 12 h. Then, the mixture was sonicated in an ice-water bath for 2 h to ensure that the nanocrystals were uniformly dispersed in the mixed solution containing the modified olefin-norbornene copolymer. Finally, the mixture was freeze-dried at -10 °C for 24 h to completely remove the cyclohexane. The product was collected to obtain the core material as a precursor for red and green fluorescent encoded microspheres. The proportion of blue and green upconversion luminescent nanocrystals was 16.7 wt%.

[0095] (4) Using a cylindrical mold, the polysulfone resin is hot-pressed at 500°C to form a cylindrical primary cladding rod with a cavity. The diameter of the primary cladding rod is 25 mm, and the diameter of the cavity inside the primary cladding rod is 2 mm. Using a cylindrical mold, the core material obtained in step (3) is hot-pressed at 230°C to form a core rod with a diameter of 2 mm. After inserting the core rod as the fiber core into the cavity of the primary cladding rod, the primary cladding rod is placed in a vacuum drying oven and heat-cured at 100°C for 30 min to obtain a primary preform. The primary preform is placed on a drawing tower and hot-drawn at 360°C into a primary fiber with a diameter of 600 μm.

[0096] (5) A secondary cladding rod with a diameter of 20 mm was made by hot pressing polysulfone resin cladding material at 300℃. A cylindrical cavity with a diameter of 6 mm was set inside the secondary cladding rod. 70 primary fibers with the same diameter and height were selected and inserted into the cavity of the secondary cladding rod. The secondary cladding rod was then placed in a vacuum drying oven and heat-cured at 100℃ for 30 min to obtain a secondary preform. The secondary preform was placed on a drawing tower and hot-drawn at 360℃ into a secondary fiber with a diameter of 600 μm.

[0097] (6) The secondary fiber is placed in a tube furnace and heat-treated at 380°C for 10 minutes to make the core of the secondary fiber reach the glass transition temperature to form a fluid and shrink into microspheres under the action of elasticity.

[0098] (7) Place the secondary fibers from step (6) into a beaker containing 50 mL of N,N-dimethylacetamide solvent. Stir continuously at 80°C until the polysulfone resin coating material is fully dissolved. Centrifuge the mixture at 13000 rpm for 5 minutes, extract the supernatant, and collect the precipitate. Add the precipitate back into the N,N-dimethylacetamide solvent and repeat the above dissolution, centrifugation, and filtration process 6 times to thoroughly remove the polysulfone resin coating material. Figure 9As shown, blue and green bicolor fluorescent coded microspheres with a particle size of 10 μm were finally obtained.

[0099] Example 7

[0100] Steps 1 and 2 in Example 7 are exactly the same as steps 1 and 2 in Example 3. The modification process and component ratio of the olefin-norbornene copolymer are the same, and will not be repeated here.

[0101] (3) Take 1g of the modified olefin-norbornene copolymer obtained in step (2) and place it in a beaker containing 35mL of cyclohexane. Heat at 60℃ and stir continuously for 40min to ensure complete dissolution of the modified olefin-norbornene copolymer. Then take 0.05g of the material with the structural formula CuSc 0.81 In 0.99 F8:0.19Er 3+ 0.01Mg 2+ Pure red upconversion luminescent nanocrystals, 0.05g with the structural formula K3Sr 3.76 In5F 26 0.2Yb, 0.02Er, 0.02Pr pure green upconversion luminescent nanocrystals and 0.15g of Ni8Sc 2.45 Al 2.55 F8:0.55Tm 3+ 0.45Mg 2+ Pure blue upconversion luminescent nanocrystals were dispersed in a container containing 1 mL of cyclohexane solvent. After the modified olefin-norbornene copolymer was completely dissolved, the solution in the container was poured into a beaker and stirred continuously for 12 h. Then, the mixture was sonicated in an ice-water bath for 2 h to uniformly disperse the nanocrystals in the mixed solution containing the modified olefin-norbornene copolymer. Finally, the mixture was freeze-dried at -30 °C for 15 h to completely remove the cyclohexane. The product was collected to obtain the core material, which served as a precursor for red, green, and blue fluorescently encoded microspheres. The proportion of red, green, and blue upconversion luminescent nanocrystals was approximately 20 wt%.

[0102] (4) Using a cylindrical mold, polysulfone resin is hot-pressed at 300°C to form a cylindrical primary cladding rod with a cavity. The diameter of the primary cladding rod is 25 mm, and the diameter of the cavity inside the primary cladding rod is 2 mm. The cavity is set along the length of the primary cladding rod. Using a cylindrical mold, the core material obtained in step (3) is hot-pressed at 230°C to form a core rod with a diameter of 2 mm. After inserting the core rod as the fiber core into the cavity of the primary cladding rod, the primary cladding rod is placed in a vacuum drying oven and heat-cured at 100°C for 30 min to obtain a primary preform. The primary preform is placed on a drawing tower and hot-drawn at 360°C into a primary fiber with a diameter of 600 μm.

[0103] (5) A secondary cladding rod with a diameter of 20 mm is made by hot pressing polysulfone resin cladding material at 300℃. A cylindrical cavity with a diameter of 6 mm is set inside the secondary cladding rod. 70 primary fibers with the same diameter and height from step (4) are selected and inserted into the cavity of the secondary cladding rod. The secondary cladding rod is then placed in a vacuum drying oven and heat-cured at 100℃ for 30 min to obtain a secondary preform. The secondary preform is then placed on a drawing tower and hot-drawn at 360℃ into a secondary fiber with a diameter of 600 μm.

[0104] (6) The secondary fiber is placed in a tube furnace and heat-treated at 380°C for 10 minutes to make the core of the secondary fiber reach the glass transition temperature to form a fluid and shrink into microspheres under the action of elasticity.

[0105] (7) Place the secondary fibers into a beaker containing 50 mL of N,N-dimethylacetamide solvent and stir continuously at 80°C until the polysulfone resin coating material is fully dissolved. Centrifuge the mixture at 13000 rpm for 5 min, extract the supernatant, and collect the precipitate. Add the precipitate back into the N,N-dimethylacetamide solvent and repeat the above dissolution, centrifugation, and filtration process 6 times to thoroughly remove the polysulfone resin coating material. Figure 10 and Figure 11 As shown, multi-color fluorescent coded microspheres with a particle size of approximately 7 μm were obtained, consisting of red, green, and blue colors.

[0106] from Figure 10 It can be observed that the red, green, and blue multicolor fluorescent encoded microspheres have good monodispersity, excellent particle size uniformity, and morphological structure.

[0107] like Figure 12 As shown, the three-dimensional matrix encoding constructed by fluorescent coded microspheres of different colors and intensities in Examples 1-7 is shown. The coordinate values ​​of the X, Y, and Z axes reflect the color signal distribution of green, blue, and red, respectively. The density of each black dot in the three-dimensional space reflects the strength of the fluorescence intensity signal. Through this method, monochromatic and multicolor fluorescent coded microspheres with ultra-large encoding capacity were obtained, which can realize fluorescent encoding for various needs.

[0108] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A method for preparing rare-earth upconversion red-green-blue fluorescently encoded microspheres, characterized in that, Includes the following steps: Step 1: Add the olefin-norbornene copolymer to a toluene solution, heat and stir at 50°C for 25-40 minutes until completely dissolved to obtain a mixed solution, and then use a coating machine to form a film from the mixed solution; Step 2: The film from Step 1 is crushed and added to bromine water. It is reacted under UV light for 12 hours, then soaked in an alkaline solution for 24 hours. The resulting solution is freeze-dried at -99°C to obtain the modified olefin-norbornene copolymer. The modified olefin-norbornene copolymer has the following structural formula: ， 1≤X≤10, 1≤Y≤10, R is a hydroxyl or amino group; Step 3: Weigh Z1wt% of the modified olefin-norbornene copolymer from Step 2 and add it to a beaker containing cyclohexane solvent. Heat and stir continuously at 60℃. Weigh Z2wt% of red upconversion luminescent nanocrystals, Z3wt% of green upconversion luminescent nanocrystals, and Z4wt% of blue upconversion luminescent nanocrystals and disperse them in a container containing cyclohexane solvent. After the modified olefin-norbornene copolymer is completely dissolved, pour the mixed solution from the container into the beaker and stir continuously for 8–12 h. After sonication in an ice-water bath for 2–3 h, freeze-dry at -30 to -10℃ for 15–24 h to obtain the core material. Wherein, 75≤Z1<100, 0≤Z2≤25, 0≤Z3≤25, 0≤Z4≤25; Z2, Z3, and Z4 are not simultaneously 0. Step 4: Hot-press the cladding material into a cylindrical primary cladding rod with a cavity. Hot-press the core material from Step 3 into a cylindrical core rod at 230°C. Place the core rod into the cavity of the primary cladding rod, and then place the primary cladding rod in a vacuum drying oven for heat curing at 100°C for 30 minutes to obtain a primary preform. Hot-draw the primary preform at 360°C to obtain primary fibers. Step 5: Hot-press the cladding material into a cylindrical secondary cladding rod with an internal cavity. Take multiple primary fibers with the same diameter and height and insert them into the cavity of the secondary cladding rod. Then place the secondary cladding rod in a vacuum drying oven and heat-cur it at 100°C for 30 minutes to obtain a secondary preform. Heat-draw the secondary preform at 360°C to obtain secondary fibers. Step 6: Heat-treat the secondary fibers from Step 5 at 350–380℃ for 3–10 minutes; Step 7: Dissolve the secondary fibers from Step 6 in the selective solvent for the coating material, heat and stir until completely dissolved, centrifuge at high speed for 5 minutes, remove the supernatant and collect the precipitate, add the precipitate back into the selective solvent for the coating material, and after repeated dissolution, centrifugation and filtration, the coating material is completely removed to obtain rare earth upconversion red-green-blue fluorescent encoded microspheres.

2. The method for preparing rare-earth upconversion red-green-blue fluorescently encoded microspheres according to claim 1, characterized in that, The structural formula of the red upconversion luminescent nanocrystals is CuSc. 0.81 In 0.99 F8:0.19Er 3+ 0.01Mg 2+ The structural formula of the green upconversion luminescent nanocrystals is K3Sr. 3.76 In5F 26 0.2Yb, 0.02Er, 0.02Pr; The structural formula of the blue upconversion luminescent nanocrystal is Ni8Sc. 2.45 Al 2.55 F8:0.55Tm 3+ 0.45Mg 2+ .

3. The method for preparing rare-earth upconversion red-green-blue fluorescently encoded microspheres according to claim 1, characterized in that, The cladding material is polysulfone resin.

4. The method for preparing rare-earth upconversion red-green-blue fluorescently encoded microspheres according to claim 3, characterized in that, The selective solvent for the cladding material is one of N,N-dimethylacetamide, N-methylpyrrolidone, N,N-dimethylformamide, or chloroform.

5. The method for preparing rare-earth upconversion red-green-blue fluorescently encoded microspheres according to claim 1, characterized in that, The alkaline solution is one of potassium hydroxide, sodium hydroxide, or ethylenediamine solution.

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