An ultrahigh quantum yield rare earth fluorescent microsphere and a preparation method thereof

Abstract

The application discloses a kind of ultra-high quantum yield rare earth fluorescent microspheres and preparation method thereof, its structure includes GdVO4 nanocrystalline core and 2-5nm YVO4 shell layer;The inner part of the YVO4 shell layer is added to the organic phase of fluorescent dye, and the dye and shell structure form nano confinement effect, and do not gather into group;Using core-shell structure rare earth nanocrystalline+high refractive index copolymer encapsulation of ultra-high quantum yield rare earth fluorescent microspheres, through the synergistic design of material structure and polymer system: make rare earth nanocrystalline adopt core enhancement doping (core: GdVO4:Eu, shell: YVO4) to improve luminous efficiency;Finally obtain the brightness of traditional fluorescent microspheres 8-20 times, quantum yield can reach 60-85% of POCT fluorescent microspheres.

Description

Technical Field

[0001] This invention relates to the field of rapid diagnostic reagent technology, and in particular to a rare earth fluorescent microsphere with ultra-high quantum yield and its preparation method. Background Technology

[0002] In the field of point-of-care testing (POCT), fluorescent microspheres are widely used as core materials for signal amplification, for example in fluorescence immunochromatography, nucleic acid lateral flow assays, and on-site screening for respiratory pathogens. Currently, mainstream fluorescent microspheres include:

[0003] The biggest pain point in the current POCT industry: (1) Fluorescent microspheres with higher luminescence intensity, lower background and better room temperature stability are required.

[0004] (2) Toxic elements (such as Cd) cannot be used. 2+ Pb 2+ This is to meet the requirements of the EU RoHS / IVD-R regulations.

[0005] (3) It needs to be compatible with the same simple immunochromatographic process as gold standard.

[0006] Existing rare earth materials exhibit strong luminescence in their powder form, but once used in microspheres: (1) Rare earth nanocrystals are prone to agglomeration, which leads to luminescence quenching; (2) The interface between the bare crystal and the aqueous phase leads to an increase in nonradiative transitions, and the quantum yield decreases to 10–30%; (3) Rare earth particles encapsulated in polymer microspheres are prone to sedimentation, leakage, and batch-to-batch variability. (4) It has high photostability but insufficient brightness, making it unable to compete with high-end fluorescence POCT; (5) The preparation process is complex (oil phase nanocrystals + emulsion phase inversion), making it difficult to mass-produce.

[0007] Therefore, there is an urgent need for rare earth fluorescent microspheres that are simple to process, have ultra-high brightness (quantum yield ≥70%), strong stability, can be industrially scaled up, and are compatible with POCT immunochromatography. Summary of the Invention

[0008] Therefore, in view of the above problems, this invention proposes a rare earth fluorescent microsphere and its preparation method that are simple to process, have ultra-high brightness (quantum yield ≥70%), strong stability, can be industrially scaled up, and are compatible with POCT immunochromatography.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: an ultra-high quantum yield rare earth fluorescent microsphere, the structure of which includes a GdVO4 nanocrystal core and a 2-5nm YVO4 shell. An organophilic fluorescent dye is added to the inner part of the YVO4 shell, and the dye forms a nano-confinence effect with the shell structure, preventing it from agglomerating. On the outside of the shell where the dye is located, a functionalized shell is covered: functional groups such as COOH, NH2, PEG, and Streptavidin.

[0010] A rare-earth fluorescent microsphere with ultra-high quantum yield can form a uniform and stable –COOH functional group layer on the surface of the microsphere by introducing a carboxylated polymer coating on the outside of the nanocrystalline shell. This functional group can react with the EDC / NHS system to generate activated esters, thereby covalently coupling with the primary amine residues of the antibody. The advantages of carboxyl surface chemistry are that the coupling conditions are mild (pH 5.5–7), the antibody activity is well preserved, it is suitable for most POCT antibody systems, and it is easy to control on a large scale.

[0011] A rare-earth fluorescent microsphere with ultra-high quantum yield is developed. Amination is achieved through polyethyleneimine coating or aminosilane grafting, forming a high-density –NH2 layer on the microsphere surface. This structure can be used for rapid cross-linking reactions mediated by glutaraldehyde, and is also suitable for forming stable covalent bonds with active esters such as NHS-biotin, thereby constructing highly efficient biotin-streptavidin systems. The advantages of amination are high reaction efficiency and fast coupling speed, making it suitable for the rapid development or construction of multivalent site binding structures.

[0012] Furthermore, the steps include: 1) Core preparation: GdVO4:Eu 3+ Hydrothermal method for preparing core nanocrystals: A suitable ligand is added to an aqueous system containing a metal ion precursor, and GdVO4 nanocrystals with uniform size and good dispersibility can be obtained through a hydrothermal reaction at 180℃ for 12 hours. The hydrothermal method has the advantages of high crystallinity, simple process, and suitability for large-scale preparation. 2) Shell coating: YVO4 shell, formed by gradually adding Y 3+ With VO4 3- The precursor can form a YVO4 shell of controllable thickness on the core surface; the 2–5 nm shell can completely cover the nanocrystal surface without significantly affecting the overall particle size or diffusion performance; the coated core-shell structure surface is more stable, less susceptible to solvent corrosion, and improves the efficiency of subsequent functionalization. During the shell growth process, organic-phase fluorescent dyes, such as Eu(TTA)3, BODIPY, and Rhodamine derivatives, are added to its inner layer, so that its molecules are firmly embedded in the shell network structure; the dye and the shell structure form a nano-confinence effect, which prevents them from agglomerating, thereby precisely fixing the dye in the inner part of the shell, i.e., the "dye-fixed sublayer"; 3) The outermost functionalized polymer / silane shell layer, outside the shell layer where the dye is located, is covered by another functionalized shell layer.

[0013] Furthermore, in step 3, carboxyl groups are introduced through silanizing reagents or carboxyl-containing molecular modifications to provide negative charge, facilitating covalent coupling with amino groups of biomolecules. Aminosilanes or amino-containing polymers are used to introduce amino groups to provide positive charge, facilitating cross-linking with carboxylated molecules for gene transfection, etc. Activated PEG is covalently coupled to the amino or carboxyl groups on the surface of nanoparticles to prolong cycle time, reduce immunogenicity, and enhance stability. The surface of nanoparticles is first functionalized, and then streptavidin is covalently coupled. Utilizing the ultra-high affinity for biotin, specific targeting or detection can be achieved. Sometimes, multiple steps may be required, such as first introducing amino groups and then connecting PEG or streptavidin.

[0014] The rare-earth fluorescent microspheres of the present invention have the following significant advantages over the prior art: (1) Ultra-high quantum yield (60–85%); (2) Brightness far exceeds that of traditional dyes and quantum dots (no toxicity issues); (3) The microsphere system has excellent stability (light resistance, heat resistance, and pH resistance); (4) It is fully compatible with POCT immunochromatographic systems and can directly replace gold standard; (5) The aqueous phase one-step method can be industrially scaled up, and the cost is lower than that of quantum dots; (6) It can be used for multiple types of IVD detection (antigen, antibody, nucleic acid capture). Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the structure of the present invention; Figure 2 This is a schematic diagram of the hierarchical structure of the present invention.

[0016] Figure 3 This is a schematic diagram of the elemental distribution of a typical rare-earth fluorescent nanosphere of the present invention.

[0017] Figure 4 The luminescence intensity of the ultra-high quantum yield rare earth fluorescent microspheres (blue columns) developed in this study was compared with that of traditional POCT fluorescent materials (gray columns, such as fluorescent microspheres, quantum dots or organic dye particles). Detailed Implementation

[0018] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments.

[0019] refer to Figures 1-4This embodiment provides an ultra-high quantum yield rare earth fluorescent microsphere and its preparation method. The structure includes a GdVO4 nanocrystal core and a 2-5 nm YVO4 shell. An organophilic fluorescent dye is added to the inner part of the YVO4 shell, and the dye forms a nano-confinence effect with the shell structure, preventing it from agglomerating. Outside the shell where the dye is located, a functionalized shell is covered with a layer of functional groups such as COOH, NH2, PEG, and Streptavidin.

[0020] This invention addresses the core-shell structure of rare-earth fluorescent microspheres by providing two types of industrially mass-producible terminal functional groups, each suitable for different coupling strategies. By adjusting the density of the surface polymer (such as PMAA, PEI, PVP, or grafted silanes), the functional group coverage can be precisely controlled, adapting it to the process requirements of various POCT products (immunochromatography, magnetic microsphere trapping, chemiluminescent substrate immobilization, etc.).

[0021] By introducing a carboxylated polymer coating on the outside of the nanocrystalline shell, a uniform and stable –COOH functional group layer can be formed on the surface of the microspheres. This functional group can react with the EDC / NHS system to generate activated esters, thereby covalently coupling with the primary amine residues of the antibody. The advantages of carboxyl surface chemistry are that the coupling conditions are mild (pH 5.5–7), the antibody activity is well preserved, it is suitable for most POCT antibody systems, and it is easy to control on a large scale.

[0022] Amination is achieved through polyethyleneimine coating or aminosilane grafting, forming a high-density –NH2 layer on the microsphere surface. This structure can be used for rapid cross-linking reactions mediated by glutaraldehyde, and is also suitable for forming stable covalent bonds with active esters such as NHS-biotin, thereby constructing highly efficient biotin-streptavidin systems. The advantages of amination are high reaction efficiency and fast coupling speed, making it suitable for the rapid development or construction of multivalent site binding structures.

[0023] By adjusting the surface polymer concentration, reaction time, and solvent conditions, the functional group density can be controlled within the range of 100–5,000 groups / µm. 2 It meets the integration requirements of different platforms such as immunochromatography, magnetic bead capture, and electrochemical sensing. Low density is suitable for monoclonal antibody directional conjugation, while high density is suitable for multivalent cross-linking and signal amplification systems. Simultaneously, this controllability ensures batch-to-batch consistency with a fluctuation of ≤5%, meeting IVD registration and mass production standards.

[0024] Product performance testing for this solution:

[0025] Figure 3This figure illustrates the elemental distribution of a typical rare-earth fluorescent nanosphere. The orange areas represent rare-earth nanocrystal nuclei (e.g., Yb / Er-doped NaYF4), which are the main luminescent centers; the green areas correspond to the encapsulated inorganic or polymer protective layers, used to isolate solvent quenching and enhance photostability; the outermost cyan-green diffusion region represents the organic fluorescent dye doped in the outer subshell, further enhancing the total fluorescence intensity through energy transfer. The hierarchical elemental distribution of the multilayer structure clearly demonstrates the core-shell-dye architecture design of the material.

[0026] Figure 4 The luminescence intensity of the ultra-high quantum yield rare-earth fluorescent microspheres (blue columns) developed in this study was compared with that of traditional POCT fluorescent materials (gray columns, such as fluorescent microspheres, quantum dots, or organic dye particles). The results show that the luminescence intensity of the nanoparticles is significantly higher than that of traditional materials, approximately 3–6 times higher, demonstrating a highly efficient coupling enhancement effect achieved through a "rare-earth core + energy-transfer shell + fluorescent dye outer layer." This improvement provides lower LOD and higher signal-to-noise ratio for POCT detection.

[0027] Furthermore, its preparation steps include: 1) Core preparation: GdVO4:Eu 3+ Hydrothermal method for preparing core nanocrystals: A suitable ligand is added to an aqueous system containing a metal ion precursor, and GdVO4 nanocrystals with uniform size and good dispersibility can be obtained through a hydrothermal reaction at 180℃ for 12 hours. The hydrothermal method has the advantages of high crystallinity, simple process, and suitability for large-scale preparation. Eu 3+ of 5 D0→ 7 The F2 energy level transition exhibits strong electric dipole emission, and the red light emission intensity can be significantly enhanced in a low-symmetry environment within the GdVO4 lattice. Doping levels of 10–20% have been confirmed in the literature as the optimal range for balancing strong luminescence and energy transfer efficiency.

[0028] Gd 3+ It has a dense energy level structure, which can serve as a medium for energy transfer, thereby enhancing Eu. 3+ The efficiency of VO4 stimulation. 3- The strong crystal field of the group helps to suppress non-radiative decay, enabling the core's internal quantum efficiency to reach a high level.

[0029] 2) Shell coating: YVO4 shell, formed by gradually adding Y 3+ With VO4 3- The precursor can form a YVO4 shell of controllable thickness on the core surface; the 2–5 nm shell can completely cover the nanocrystal surface without significantly affecting the overall particle size or diffusion performance; the coated core-shell structure surface is more stable, less susceptible to solvent corrosion, and improves the efficiency of subsequent functionalization. The primary energy loss in rare-earth nanocrystals comes from vibrational quenching caused by surface defects and hydroxyl groups. The YVO4 shell forms a physical barrier, effectively blocking high-frequency vibrational coupling and increasing the external quantum yield from 50–60% to ≥85%.

[0030] The coated core-shell structure exhibits greater surface stability, is less susceptible to solvent corrosion, and enhances the efficiency of subsequent functionalization processes (such as carboxylation, amination, and hydrophilic coating). This structure is more suitable for applications in aqueous systems such as point-of-care testing (POCT).

[0031] During the growth of the shell, an organic-phase fluorescent dye, such as Eu(TTA)3, BODIPY, or Rhodamine derivative, is added to the inner layer, so that its molecules are firmly embedded in the shell network structure. The dye and the shell structure form a nano-confinence effect, which prevents them from aggregating into clusters, thereby fixing the dye precisely in the inner part of the shell, i.e., the "dye-fixed sublayer". The dye and shell structure form a nano-confinement effect, preventing aggregation and thus avoiding common concentration quenching. This fixed position, 5–10 nm away from the rare-earth core, effectively utilizes Förster or Dexter-type energy transfer to achieve a composite amplification effect of "rare-earth core excitation + organic dye-enhanced luminescence." This structure ensures the dye is neither exposed to external elements nor loses fluorescence due to accumulation, significantly improving quantum yield.

[0032] 3) The outermost functionalized polymer / silane shell layer, outside the shell layer where the dye is located, is covered by another functionalized shell layer.

[0033] The outer shell serves three purposes: ① Completely seal the dye layer → prevent dye leakage and ensure that the dye remains stable and does not fall off even at 37℃ in a protein-containing environment.

[0034] ② Avoid direct exposure of dye to the aqueous phase → significantly reduce solvent quenching and improve luminescence lifetime.

[0035] ③ Provides coupling sites → can be covalently linked with biomolecules such as antibodies, oligonucleotides, and antigens for use in point-of-care testing (POCT).

[0036] Because the dye is deeply embedded inside the double shell, its stability is 5–10 times better than that of traditional self-fluorescent microspheres.

[0037] Furthermore, in step 3, carboxyl groups are introduced through silanizing reagents or carboxyl-containing molecular modifications to provide negative charge, facilitating covalent coupling with amino groups of biomolecules. Aminosilanes or amino-containing polymers are used to introduce amino groups to provide positive charge, facilitating cross-linking with carboxylated molecules for gene transfection, etc. Activated PEG is covalently coupled to the amino or carboxyl groups on the surface of nanoparticles to prolong cycle time, reduce immunogenicity, and enhance stability. The surface of nanoparticles is first functionalized, and then streptavidin is covalently coupled. Utilizing the ultra-high affinity for biotin, specific targeting or detection can be achieved. Sometimes, multiple steps may be required, such as first introducing amino groups and then connecting PEG or streptavidin.

[0038] Hydrophilic treatment of nanocrystalline surfaces: Currently, most rare earth crystals are oil-phase ligands (OA / OM), which are not directly compatible with POCT processes. This invention employs a sulfonate grafting + polyetherification modification method:

[0039] The treated nanocrystals remained stably dispersed in the aqueous phase for more than 6 months.

[0040] The stability tests of the microsphere system are as follows (light resistance, heat resistance, pH resistance). Photostability test (continuous illumination for 12 hours)

[0041] Rare earth microspheres show almost no photobleaching under high-intensity excitation light, with a loss of only 8%, which is far superior to traditional organic dyes (which suffer severe photobleaching) and some quantum dots (which experience fluorescence decay due to surface oxidation).

[0042] Heat resistance test (treated at 37–60℃ for 1 hour)

[0043] Rare earth core-shell structures exhibit excellent thermal stability, maintaining >94% luminescence even at 55–60℃; traditional dye-type microspheres show significant fluorescence decay due to polymer softening and dye liquefaction and migration.

[0044] pH stability test (pH 4–10)

[0045] Rare earth microspheres are almost unaffected within the pH range of 4–10, maintaining >93% luminescence; traditional fluorescent microspheres are sensitive to both acids and bases, exhibiting dye leakage or fluorescence quenching.

[0046] Stability in high-salt and protein environments (for POCT scenarios)

[0047] Rare earth microspheres remained stable under high salt, protein, and serum conditions, demonstrating that the outer functionalized shell effectively prevented protein adsorption and dye leakage.

[0048] Accelerated aging test (placed at 37°C for 7 days)

[0049] This invention proposes an ultra-high quantum yield rare-earth fluorescent microsphere consisting of "core-shell structured rare-earth nanocrystals encapsulated with high-refractive-index copolymers," achieved through the synergistic design of the material structure and polymer system: (1) Rare earth nanocrystals employ core-enhanced doping (core: GdVO4:Eu, shell: YVO4) to improve luminescence efficiency; (2) The shell isolates the surface solvent quenching, so that the quantum yield of the powder is maintained in the microsphere; (3) The microsphere matrix is ​​composed of a high refractive index polymer, which improves the light emission efficiency; (4) Introduce carboxyl or amino groups on the surface to achieve antibody conjugation; (5) Aqueous phase one-step particle encapsulation technology ensures that rare earth nanocrystals are uniformly dispersed and do not agglomerate; The final result is fluorescent microspheres for POCT with a brightness 8–20 times that of traditional fluorescent microspheres and a quantum yield of 60–85%.

[0050] This material can be used for: (1) Fluorescent immunochromatography (an upgraded alternative to FITC / Cy5); (2) Rapid diagnosis using viral antigens; (3) Tumor markers POCT (CEA, AFP, CHI3L1, suPAR, etc.); (4) POCT of myocardial markers (cTnI, NT-proBNP).

[0051] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered illustrative and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0052] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A rare-earth fluorescent microsphere with ultra-high quantum yield, characterized in that: Its structure includes a GdVO4 nanocrystalline core and a 2-5 nm YVO4 shell; The inner layer of the YVO4 shell contains an organophilic fluorescent dye, and the dye forms a nano-confinence effect with the shell structure, preventing it from agglomerating. Outside the dye shell, there is another functionalized shell: functional groups such as COOH, NH2, PEG, and Streptavidin.

2. A rare-earth fluorescent microsphere with ultra-high quantum yield, characterized in that: By introducing a carboxylated polymer coating on the outside of the nanocrystalline shell, a uniform and stable –COOH functional group layer can be formed on the surface of the microspheres. This functional group can react with the EDC / NHS system to generate activated esters, thereby covalently coupling with the primary amine residues of the antibody. The advantages of carboxylated surface chemistry are that the coupling conditions are mild: pH 5.5–7.4, the antibody activity is well preserved, it is suitable for most POCT antibody systems, and it is easy to control on a large scale.

3. A rare-earth fluorescent microsphere with ultra-high quantum yield, characterized in that: Amination is achieved through polyethyleneimine coating or aminosilane grafting, forming a high-density –NH2 layer on the microsphere surface. This structure can be used for rapid cross-linking reactions mediated by glutaraldehyde, and is also suitable for forming stable covalent bonds with active esters such as NHS-biotin, thereby constructing highly efficient biotin-streptavidin systems. The advantages of amination are high reaction efficiency and fast coupling speed, making it suitable for rapid development (R&D stage) or the construction of multivalent binding structures.

4. The method for preparing ultra-high quantum yield rare earth fluorescent microspheres according to claim 1, characterized in that: The steps include: 1) Core preparation: GdVO4:Eu 3+ (17%) Hydrothermal preparation of core nanocrystals: By adding an appropriate amount of ligand to an aqueous system containing a metal ion precursor, GdVO4 nanocrystals with uniform size and good dispersibility can be obtained through a hydrothermal reaction at 180℃ for 12 hours. The hydrothermal method has advantages such as high crystallinity, simple process, and suitability for large-scale preparation. 2) Shell coating: YVO4 shell; By gradually adding Y 3+ With VO4 3- The precursor can form a YVO4 shell of controllable thickness on the core surface. The 2–5 nm shell can completely cover the nanocrystal surface without significantly affecting the overall particle size or diffusion performance; the coated core-shell structure has a more stable surface, is less susceptible to solvent corrosion, and improves the efficiency of subsequent functionalization. During the growth of the shell, an organic fluorescent dye, such as Eu(TTA)3, BODIPY, or Rhodamine derivative, is added to the inner layer, so that its molecules are firmly embedded in the shell network structure. The dye and the shell structure form a nano-confinence effect, which prevents them from agglomerating, thereby fixing the dye precisely in the inner part of the shell, i.e., the "dye-fixed sublayer". 3) Outermost functionalized polymer / silane shell; Outside the dye shell, a functionalized shell (COOH, NH2, PEG, Streptavidin, etc.) is then covered.

5. The method for preparing ultra-high quantum yield rare earth fluorescent microspheres according to claim 1, characterized in that: In step 3, a carboxyl group is introduced through a silanizing agent or a carboxyl-containing molecular modification to provide negative charge, which facilitates covalent coupling with the amino groups of biomolecules. The amino group is introduced by treating with aminosilanes or amino-containing polymers to provide positive charge, which facilitates cross-linking with carboxylated molecules and is used for gene transfection, etc. Activated PEG is covalently coupled to the amino or carboxyl groups on the surface of nanoparticles to prolong cycling time, reduce immunogenicity, and enhance stability. First, the surface of nanoparticles is functionalized (e.g., by introducing carboxyl / amino groups), and then streptavidin is covalently coupled to achieve specific targeting or detection by utilizing its ultra-high affinity for biotin. Sometimes a multi-step reaction may be required, such as first introducing an amino group and then linking it with PEG or streptavidin.

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