A fluorescent probe based on ceramic fluorescent composite particles, and a preparation method and application thereof

Abstract

This invention relates to a fluorescent probe based on ceramic fluorescent composite particles, its preparation method, and its application. The ceramic fluorescent composite particles comprise multiple fluorescent nanocrystals and an inorganic material; the inorganic matrix is ​​derived from a mesoporous material, and the multiple fluorescent nanocrystals are dispersed within the pore structure of the mesoporous material; the ceramic fluorescent composite particles undergo sintering, causing the framework of the inorganic matrix to shrink and densify, resulting in partial or complete closure of the pore structure, thereby forming a dense coating layer on the surface of the fluorescent nanocrystals; the particle size of the ceramic fluorescent composite particles is 50–300 nm, and the density is 1.1 g / cm³. 3 Up to 6 g / cm 3 The fluorescent nanocrystals are loaded into the total mass of the ceramic fluorescent composite particles at a rate of 10 wt% to 35 wt%; the absolute quantum yield of the ceramic fluorescent composite particles is above 85%.

Description

Technical Field

[0001] This invention belongs to the field of materials chemistry, specifically relating to a fluorescent probe based on ceramic fluorescent composite particles, its preparation method, and its application. Background Technology

[0002] Fluorescence immunochromatography (FIC) boasts advantages such as high sensitivity, high accuracy, strong quantitative capability, and good practicality, making it widely used in medical diagnostic fields such as infectious disease screening and tumor marker diagnosis. It serves as a powerful complement to traditional detection methods and large-scale instrument-based detection. However, the samples involved in FIC are often quite complex, placing extremely high demands on the stability and fluorescence performance of the fluorescent probe materials.

[0003] Fluorescent nanocrystals possess advantages such as high quantum yield, narrow emission spectrum, tunable emission color, and high color purity, making them effective tools in medical diagnostics and widely used in biomedical fields, including fluorescence immunochromatography, cell, tissue, and in vivo imaging. However, traditional fluorescent nanocrystals are mostly prepared via solution methods, resulting in materials with poor stability, easily decomposed or quenched by light, heat, moisture, and oxygen. Furthermore, some fluorescent nanocrystals may contain heavy metals such as cadmium and lead, posing potential biotoxicity risks; moreover, fluorescent nanocrystals are prone to aggregation. These factors severely limit their application prospects in the biomedical field.

[0004] Constructing a core-shell structure to protect fluorescent nanocrystals is an effective strategy to enhance their stability and prevent the leakage of toxic heavy metals and the self-aggregation of nanocrystals. Existing coating strategies mainly include liquid-phase coating and inorganic mesoporous material coating. However, the shells prepared by liquid-phase coating are usually relatively porous and cannot completely prevent moisture or oxygen from corroding the fluorescent nanocrystals. Especially in complex physiological environments containing urine, saliva, and blood, the stability of such liquid-phase coated fluorescent nanocrystals still fails to meet the requirements of practical applications. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a fluorescent probe based on ceramic fluorescent composite particles, its preparation method, and its application.

[0006] In a first aspect, the present invention provides a ceramic fluorescent composite particle with a sintered confined structure, the ceramic fluorescent composite particle comprising a plurality of fluorescent nanocrystals and an inorganic material; the inorganic matrix is ​​derived from a mesoporous material, the plurality of fluorescent nanocrystals being dispersed in the pore structure of the mesoporous material; the ceramic fluorescent composite particle is obtained by sintering, during which the skeleton of the inorganic matrix shrinks and densifies, causing the pore structure to partially or completely close, thereby forming a densified coating layer on the surface of the fluorescent nanocrystals.

[0007] Preferably, the ceramic fluorescent composite particles have a particle size of 50-300 nm and a density of 1.1 g / cm³. 3 Up to 6 g / cm 3 The fluorescent nanocrystals are loaded into the total mass of the ceramic fluorescent composite particles at a rate of 10 wt% to 35 wt%. The absolute quantum yield of the ceramic fluorescent composite particles is above 85%.

[0008] More preferably, the loading of the fluorescent nanocrystals is 25 wt% to 30 wt%.

[0009] In some embodiments, the mesoporous material is selected from at least one of mesoporous single oxides or mesoporous composite oxides; The mesoporous single oxide includes at least one of mesoporous silica, mesoporous alumina and mesoporous titanium dioxide; The mesoporous composite oxide includes mesoporous silicon dioxide-metal oxide.

[0010] In some embodiments, the structure of the fluorescent nanocrystals is selected from any of the following: (1) The perovskite structure has a stoichiometric ratio of ABX3; where B is selected from Sn, Pb, Cu, Mn, Ca, Sr, Ba or a combination thereof; (2) Non-perovskite structure, the stoichiometric ratio of which is selected from ABX3, A4BX6 or A2BX5; wherein B is selected from Pb, Zn, Ca, Ba or a combination thereof; In any of the above structures, A is independently selected from Rb, K, or Cs; X is independently selected from F, Cl, Br, or I.

[0011] In some embodiments, the sintering temperature is 400-900°C and the time is 2.5-5 hours.

[0012] In some embodiments, the mesoporous silica has a particle size of 20-600 nm and a pore size of 2-15 nm, and the sintering temperature of the ceramic fluorescent composite particles with the mesoporous silica as the framework is 400-600℃. The loading of fluorescent nanocrystals in the fluorescent composite particles is higher than 20%wt, and the absolute quantum yield of the ceramic fluorescent composite particles is above 90%.

[0013] In some embodiments, the mesoporous silica-metal composite is mesoporous silica-alumina, and the sintering temperature of the fluorescent composite particles with the mesoporous silica-alumina as the framework is 600-900℃, so that the coating layer (shell) forms a dense aluminosilicate structure, which significantly improves the fluorescence retention rate of the ceramic fluorescent composite particles in a water-oxygen environment.

[0014] In some embodiments, the fluorescent nanocrystals have a CsPb structure. 1-x Sr x Br3 in which 0 <x<1。

[0015] Preferably, prior to the sintering process, the fluorescent composite particles are further subjected to a drying process at a temperature of 80-100°C.

[0016] Secondly, the present invention provides a method for preparing the ceramic fluorescent composite particles, comprising the following steps: S1. Cesium salt and metal salt are added to an aqueous medium and ultrasonically treated at room temperature to form a dispersion, wherein the metal salt is lead salt and / or strontium salt; S2. Add mesoporous silica or mesoporous silica / alumina to the dispersion, stir and evaporate at 80-100℃ until the water evaporates completely to obtain a solid mixture; then sinter the solid mixture at 400-900℃ for 2.5-5h to obtain the ceramic fluorescent composite particles.

[0017] In some embodiments, step S1 of the preparation method involves adding CsBr and PbBr2 to water and ultrasonically treating them at room temperature to form a dispersion, wherein the molar ratio of CsBr to PbBr2 is 1:(0.9-1.8), and the ultrasonic treatment time is 4-8 minutes.

[0018] Thirdly, the present invention provides the use of the ceramic fluorescent composite particles, the use including using the ceramic fluorescent composite particles for: (a1) Prepare fluorescent immunochromatographic test strips or kits; (a2) Prepare fluorescent probes for in vitro cell imaging or in vivo fluorescence imaging; (a3) Prepare drug delivery carriers or drug controlled release systems.

[0019] This invention provides a fluorescent probe, the fluorescent probe comprising: The core particles comprise ceramic fluorescent composite particles as described above; A surface modification layer that covers the surface of the core particles; And biorecognition molecules (such as proteins) that are fixed on the surface modification layer; The surface modification layer includes a silane coupling agent-derived layer, and optionally, a polymer coating layer. The silane coupling agent-derived layer and the polymer coating layer provide reactive groups, which are covalently linked to the biorecognition molecule.

[0020] Preferably, the reactive group is selected from at least one of carboxyl, thiol, amino, epoxy, aldehyde or maleimide.

[0021] In some embodiments, the surface modification layer includes: an aminosilane underlayer (e.g., formed of APTES) covering the surface of the core particles; and a carboxyl linker connected to the aminosilane underlayer via an amide bond, the carboxyl linker being derived from the ring-opening reaction of succinic anhydride; the biorecognition molecule is immobilized by a dehydration condensation reaction with the carboxyl group at the end of the carboxyl linker.

[0022] In some embodiments, when the reactive group is a carboxyl group, the resulting carboxyl-functionalized ceramic fluorescent composite particles have a hydrodynamic average particle size of 50~500 nm and a polydispersity index of less than 0.2 in the aqueous phase.

[0023] In some embodiments, the fluorescent probe is prepared from highly monodisperse surface-carboxylated ceramic fluorescent composite particles as a precursor; wherein, the surface-carboxylated ceramic fluorescent composite particles (i.e., the intermediate before coupling with the biorecognition protein) preferably have a hydrodynamic average particle size of 250 nm - 290 nm in the aqueous phase, a polydispersity index (PDI) of less than 0.1, and a surface zeta potential (pH 7.0) of -15 mV to -60 mV.

[0024] This invention provides a method for preparing a fluorescent probe, comprising the following steps: S1: Surface modification treatment is performed on the ceramic fluorescent composite particles to obtain surface-functionalized ceramic fluorescent composite particles; the surface modification treatment includes at least one of the following methods: (a) Silanization-derivation modification: The ceramic fluorescent composite particles are contacted with a silane coupling agent containing an anhydride group and then hydrolyzed; or; The ceramic fluorescent composite particles are first contacted with an amino-containing silane coupling agent and then ring-opening reaction is carried out with an anhydride compound. (b) Silane-polymer coupling modification: First, the ceramic fluorescent composite particles are contacted with an amino-containing silane coupling agent to introduce surface amino groups; then, under the action of an activator, the surface amino groups undergo a condensation reaction with a carboxyl-containing polymer. (c) Surface in-situ polymerization modification: First, the ceramic fluorescent composite particles are contacted with a silane coupling agent containing polymerizable active groups to introduce surface active sites; then, under the action of an initiator, the carboxyl-containing monomers are grafted and polymerized on the surface of the particles. S2. Disperse the surface-functionalized ceramic fluorescent composite particles in a buffer solution with a pH of 5.0-9.5, add a cross-linking agent and protein, and incubate to obtain a fluorescent probe of surface-modified protein.

[0025] In some embodiments, the amino-containing silane coupling agent is selected from one or more of 3-aminopropyltriethoxysilane, diethylenetriaminopropyltrimethoxysilane, 3-(methacryloyloxy)propyltrimethoxysilane, 3-aminopropyltrimethoxysilane, N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, N-(2-aminoethyl)-3-aminopropyltriethoxysilane, 3-ureapropyltriethoxysilane, 3-methacryloyloxypropyltriethoxysilane, 3-acryloyloxypropyltrimethoxysilane, vinyltriethoxysilane, or vinyltrimethoxysilane; and / or; The silane coupling agent containing anhydride groups is selected from [3-(trimethoxysilyl)propyl]succinic anhydride, 3-(triethoxysilyl)propylsuccinic anhydride; and / or; The anhydride compound is selected from at least one of succinic anhydride, glutaric anhydride, maleic anhydride, phthalic anhydride, trimellitic anhydride, or itaconic anhydride; and / or; The initiator is selected from at least one of ammonium persulfate, potassium persulfate, azobisisobutyronitrile, benzoyl peroxide, and 2,2'-azobis(2-methylpropionitrile); and / or; The carboxyl-containing polymer is selected from at least one of polyacrylic acid, polymethacrylic acid, polyglutamic acid, polyaspartic acid, carboxymethyl cellulose, alginate, or their salts.

[0026] In some embodiments, the carboxyl-containing monomer is selected from one or more of acrylic acid, methacrylic acid, carboxylic betaine, polyethylene glycol diacrylate, N-vinylpyrrolidone, and sulfonate betaine.

[0027] In some embodiments, the buffer solution is one or more of morpholine ethanesulfonic acid buffer, phosphate buffer, borax-boric acid buffer, tris(hydroxymethyl)aminomethane hydrochloride buffer, sodium barbital-hydrochloric acid buffer, borax-sodium hydroxide, glycine-sodium hydroxide, and sodium carbonate-sodium bicarbonate; and / or the pH of the buffer solution is 4 to 10.

[0028] In some embodiments, the activator is one or more of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, sodium N-hydroxysuccinimide sulfonate, sodium 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester, N,N'-dicyclohexylcarbodiimide, and 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester.

[0029] In some embodiments, the mass ratio of the amount of crosslinking agent added to the amount of surface-functionalized ceramic fluorescent composite particles is 1:1 to 100:1.

[0030] In some embodiments, the protein includes respiratory syncytial virus antibody, influenza A virus antibody, influenza B virus antibody, and human chorionic gonadotropin antibody.

[0031] In some embodiments, the mass ratio of the amount of surface-functionalized ceramic fluorescent composite particles added to the amount of protein added is 1:10 to 50:1.

[0032] In some embodiments, the incubation temperature is 4~40°C.

[0033] In some embodiments, the preparation method of the fluorescent probe includes the following steps: (1) 3-aminopropyltriethoxysilane (APTES) was dispersed in anhydrous ethanol, ceramic fluorescent composite particles were added, and the reaction was carried out under heating and stirring conditions to obtain composite particles with amino functionalization on the surface. (2) The surface amino-functionalized composite particles are dispersed in N,N-dimethylformamide, and succinic anhydride is added to carry out a ring-opening reaction to obtain surface carboxyl-functionalized composite particles. (3) The surface carboxyl-functionalized composite particles are dispersed in a weakly acidic buffer solution, the surface carboxyl groups are activated with an activator, and then a specific protein is added for incubation to form a covalent link; (4) After cleaning, the unreacted sites are blocked with a blocking agent and the fluorescent probe is obtained after cleaning.

[0034] The specific protein is a polyclonal antibody against human respiratory syncytial virus.

[0035] In some embodiments, the concentration of 3-aminopropyltriethoxysilane (APTES) in anhydrous ethanol in step (1) is 4-6 mM. The concentration of the ceramic fluorescent composite particles in the reaction system is 1 mg / mL - 1.5 mg / mL; and the reaction temperature is 50℃-70℃, and the reaction time is 1.5-2.5 hours.

[0036] In some embodiments, the mass ratio of succinic anhydride to surface amino-functionalized ceramic fluorescent composite particles in step (2) is (2-3):1; the reaction is carried out at room temperature and stirred overnight.

[0037] In some embodiments, the weakly acidic buffer in step (3) is a morpholine ethanesulfonic acid buffer with a pH of 5.0-6.5; the activator is 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) or a mixture of EDC and N-hydroxysuccinimide (NHS); and the activation time is 15-45 minutes.

[0038] In some embodiments, the incubation temperature is 4~40°C.

[0039] In summary, compared with the prior art, this application achieves the following technical effects: This invention constructs a dense silica / metal oxide shell followed by a tight polymer / silane modification layer, forming a dual physical barrier around the luminescent center (quantum dot). This dense structure effectively blocks the penetration and erosion of water molecules, oxygen, and acid / base ions, thus solving the problem of structural collapse or ion leaching that traditional quantum dots are prone to in aqueous buffer solutions or complex biological samples (such as acidic or alkaline environments), significantly extending the shelf life of the material.

[0040] The composite particles of this invention achieve effective encapsulation and protection of the internal light-emitting center by constructing a dense shell structure, which significantly reduces the erosion and interference of environmental factors on the light-emitting center, thereby endowing the composite particles with excellent anti-photobleaching properties (photostability).

[0041] Under long-term continuous irradiation with high-intensity excitation light, the composite particles of the present invention can effectively resist photo-oxidation and the fluorescence intensity does not decrease significantly. This overcomes the defect that traditional organic dyes or exposed quantum dots are prone to photobleaching and meets the needs of long-term bioimaging or high-sensitivity detection.

[0042] Unlike existing technologies that commonly employ physical adsorption or dynamic ligand exchange strategies, this invention introduces surface functional groups (such as carboxyl groups) through in-situ polymerization / covalent grafting. These covalently anchored functional ligands exhibit extremely high structural stability, preventing ligand detachment or dissociation during dilution, washing, or long-term storage. This not only ensures the colloidal stability of the particles but also provides stable and abundant reaction sites for subsequent coupling with biomolecules (such as antibodies and proteins), significantly improving coupling efficiency and detection reproducibility.

[0043] Unlike existing fluorescent microspheres which generally suffer from insufficient fluorescent nanocrystal loading capacity, this invention significantly improves the loading capacity of microspheres for fluorescent nanocrystals while maintaining high quantum fluorescence yield by introducing the synergistic effect of specific metal ion doping of fluorescent nanocrystals and low-temperature sintering process. On the one hand, the physical isolation effect of mesoporous materials on fluorescent nanocrystals effectively suppresses the aggregation-induced quenching (ACQ) effect at high concentrations. On the other hand, the coordination effect of metal ions repairs the generation of halogen vacancies in nanocrystals. Thanks to the synergistic effect of defect passivation and spatial isolation, the fluorescent microspheres can maintain excellent absolute quantum fluorescence efficiency while achieving high loading capacity.

[0044] The preparation process of this invention effectively passivates the defect states on the surface of quantum dots and achieves high-density quantum dot loading while preventing quantum dot aggregation and quenching. The resulting composite particles exhibit high fluorescence quantum yield and strong fluorescence emission intensity, which can significantly improve the signal-to-noise ratio in applications such as immunochromatography, enabling ultrasensitive detection of low-concentration target analytes. Attached Figure Description

[0045] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0046] Figure 1 This is the preparation process flow of the ceramic fluorescent probe described in this invention.

[0047] Figure 2 The adsorption-desorption isotherms of mesoporous silica before and after sintering in Example 1 are shown.

[0048] Figure 3 This is an optical photograph of the ceramic fluorescent composite particles prepared in Example 1 of this invention.

[0049] Figure 4 This is an optical photograph of the amino-functionalized ceramic fluorescent composite particles prepared in Example 1 of this invention.

[0050] Figure 5 This is a transmission electron microscope (TEM) image of the ceramic fluorescent composite particles prepared in Example 1 of this invention.

[0051] Figure 6 This is the X-ray diffraction (XRD) pattern of the ceramic fluorescent composite particles prepared in Example 1 of this invention, and a comparison diagram with the standard PDF card.

[0052] Figure 7The image shows a comparison of the FT-IR spectra of the surface-carboxylated ceramic fluorescent composite particles obtained in step (4) of Example 1 of the present invention and the surface-carboxylated ceramic fluorescent composite particles obtained in step (2).

[0053] Figure 8 The results are DLS characterization of the surface carboxylated ceramic fluorescent composite particles in Example 1 of this invention.

[0054] Figure 9 The Zeta potential characterization results are for the surface carboxylated ceramic fluorescent composite particles in Example 1 of this invention.

[0055] Figure 10 The energy dispersive X-ray spectrum of the high-fluorescence nanocrystal loading ceramic fluorescent composite particles in Example 2 of this invention is shown.

[0056] Figure 11 The absolute quantum yield test spectrum of the ceramic fluorescent composite particles with high fluorescent nanocrystal loading in Example 2 of the present invention is shown.

[0057] Figure 12 The absolute quantum yield test spectrum of ceramic fluorescent composite particles with high fluorescent nanocrystal loading that did not use the Sr metal doping process in Example 2 of this invention.

[0058] Figure 13 This is a schematic diagram of the structure of the fluorescent immunochromatographic test strip described in this invention.

[0059] Figure 14 This is an optical photograph of the respiratory syncytial virus ceramic fluorescent probe test strip prepared in Example 3 of the present invention.

[0060] Figure 15 This is a schematic diagram of the linear range of the respiratory syncytial virus ceramic fluorescent probe test strip prepared in Example 3 of the present invention.

[0061] Figure 16 Optical photographs taken under ultraviolet light before and after surface modification of silica-coated CsPbBr3 core-shell nanocrystals prepared by conventional liquid-phase method in Comparative Example 1 of this invention.

[0062] Figure 17 These are results from laser scanning confocal microscopy. Detailed Implementation

[0063] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0064] This invention provides a fluorescent probe based on ceramic fluorescent composite particles, its preparation method, and its application.

[0065] Figure 1 This invention demonstrates a method for preparing fluorescent probes based on ceramic fluorescent composite particles. The main idea is to load multiple fluorescent nanocrystals onto an inorganic mesoporous material, and then sinter them at high temperature. This causes the pores of the inorganic mesoporous material to collapse, achieving a tight encapsulation of the fluorescent nanocrystals by an oxide layer, resulting in ceramic fluorescent composite particles. These particles are then encapsulated with a silane coupling agent or polymer to obtain surface-functionalized ceramic fluorescent composite particles. Finally, these surface-functionalized ceramic fluorescent composite particles are co-incubated with proteins in a cross-linking buffer to obtain the ceramic fluorescent probe. The ceramic fluorescent composite particles significantly improve the stability of the fluorescent probe, further enhancing the sensitivity, accuracy, and specificity of fluorescence immunochromatography for complex samples.

[0066] The ceramic fluorescent composite particles prepared in the embodiments of the present invention have a particle size of 50~300nm and a density of 1.1 g / cm³. 3 Up to 6g / cm 3 Ceramic fluorescent composite particles within this size range exhibit excellent solution processing properties and fluorescence characteristics. Furthermore, these particles are particularly suitable for bioassay applications, effectively increasing the binding amount / labeling density of probes on the surface of biological samples, thereby enhancing the detection signal.

[0067] When implementing the technical solution of this invention, those skilled in the art can make adjustments according to the actual situation, specifically including: 1. It is necessary to select inorganic mesoporous materials with appropriate pore sizes according to the type of nanocrystals so that fluorescent nanocrystals can be filled into inorganic mesoporous materials; 2. The particle size, type, and composition of the nanocrystalline precursor of the inorganic mesoporous material need to be selected according to the molecular weight of different test samples and antibodies to ensure the high antibody loading of the ceramic fluorescent composite particles and the high sensitivity and accuracy of the ceramic fluorescent probe when it is further applied to fluorescence immunochromatography. 3. The high-temperature sealing temperature needs to be determined according to the type of inorganic mesoporous material to avoid excessive agglomeration of the internal material at high temperatures; 4. Different silane coupling agents and surface functionalization methods need to be selected according to different cross-linking agents to ensure that the protein and ceramic fluorescent composite particles can be covalently bound. 5. Different proteins need to be selected for covalent coupling according to different medical diagnostic projects to ensure the high specificity of the fluorescent probe for further application in fluorescence immunochromatography.

[0068] The materials included in the embodiment are: F127, also known as Pluronic F127 or Poloxamer 407, CAS Registry Number 9003-11-6, is a nonionic block copolymer synthesized from propylene oxide, propylene glycol, and ethylene oxide.

[0069] TEOS, short for Tetraethyl orthosilicate, is chemically named Tetraethoxysilane and is also commonly known as Ethyl silicate. Its CAS Registry Number is 78-10-4, and its molecular weight is 208.33 g / mol.

[0070] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used are commercially available.

[0071] Example 1: Preparation of Ceramic Fluorescent Probes for Respiratory Syncytial Virus A method for preparing a respiratory syncytial virus ceramic fluorescent probe, the specific steps of which are as follows: (1) Preparation of mesoporous silica: Add 40 mL of deionized water to a 100 mL beaker, followed by 1 g of hexadecyltrimethylammonium chloride (CTAC) and 0.2 g of triethanolamine (TEA). Stir the mixture at 500 rpm for 1 h at 70 °C until the solution becomes clear and transparent. Then add 3 mL of tetraethyl orthosilicate (TEOS), and continue stirring at 70 °C for 1 h, then stop the reaction. After the reaction is complete, stop heating, and after the solution returns to room temperature, centrifuge to collect the precipitate, and dry it at 80 °C. Grind the dried powder, add 150 mL of isopropanol, reflux overnight to release the mold, and prepare mesoporous silica. This step can obtain mesoporous silica with a particle size of 200-400 nm and a pore size of 2-4 nm, which has good monodispersity and uniform particle size.

[0072] (2) Preparation of silica-dense coated CsPbBr3 fluorescent composite particles: Add 0.3 mmol cesium bromide, 0.5 mmol lead bromide and 100 mL water to a 100 mL beaker, ultrasonically disperse at room temperature for 5 minutes, then add 0.35 g of the above mesoporous silica, stir at 90 °C until the mixture is dry, and sinter at 500 °C for 3 h to obtain ceramic fluorescent composite particles.

[0073] Figure 2 The adsorption-desorption isotherms are for mesoporous silica before and after sintering. Figure 2 The result shows that the specific surface area (BET) of mesoporous silica after sintering increased from 739.556 m². 2 / g dropped to 13.541 m 2 / g, proved that the mesoporous structure collapsed and a dense silica surface was formed.

[0074] (3) Preparation of surface amino-functionalized ceramic fluorescent composite particles: 50 μL of 3-aminopropyltriethoxysilane (APTES) was dispersed in 40 mL of anhydrous ethanol, and then 50 mg of the above ceramic fluorescent composite particles were added. The mixture was stirred at 60 °C for 2 h to obtain surface amino-functionalized ceramic fluorescent composite particles.

[0075] (4) Preparation of surface carboxyl-functionalized ceramic fluorescent composite particles: The above surface amino-functionalized ceramic fluorescent composite particles were dispersed in 20 mL of N,N-dimethylformamide (DMF), and 20 mg of succinic anhydride was added. The mixture was stirred overnight.

[0076] (5) Preparation of respiratory syncytial virus fluorescent probe: 100 μg of the above surface carboxyl-functionalized ceramic fluorescent composite particles were dispersed in 500 μL morpholine ethanesulfonic acid buffer (50 mM, pH=6.0), 100 μg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide was added, and the mixture was incubated at room temperature for 30 minutes. Then, 10 μg of rabbit anti-human respiratory syncytial virus polyclonal antibody was added, and the mixture was incubated at 4°C overnight. After washing, the mixture was blocked with 5% bovine serum albumin for 30 minutes and washed again to obtain the respiratory syncytial virus ceramic fluorescent probe.

[0077] Test Example 1: Morphology, structure and luminescence properties of the ceramic fluorescent composite particles obtained in steps (2) and (3) of Example 1 (1) Appearance characteristics Figure 3 The image shows a fluorescence optical photograph of the ceramic fluorescent composite particles sintered at high temperature in step (2) of Example 1 of this invention. Figure 3 As shown, the material is a uniformly distributed bright yellow powder.

[0078] Figure 4The image shows a fluorescence optical photograph of the amino-functionalized ceramic fluorescent composite particles obtained in step (3) of Example 1 of the present invention, which shows strong fluorescence emission.

[0079] (2) Microstructure Figure 5 This is a transmission electron microscope (TEM) image of the ceramic fluorescent composite particles obtained in step (2) of Example 1 of the present invention. The image shows that the particles are uniform in size, with a diameter of approximately 150-250 nm, and the particle boundaries are clear and the dispersion is good.

[0080] (3) Crystal structure Figure 6 The XRD pattern and corresponding PDF card of the ceramic fluorescent composite particles obtained in step (2) of Example 1 of this invention are shown. XRD analysis shows that the characteristic diffraction peaks of CsPbBr3 fluorescent nanocrystals in the composite material are in high agreement with the corresponding standard PDF card (#97-002-9073), proving that CsPbBr3 fluorescent nanocrystals have successfully crystallized in the silica coating layer and maintained a good crystal structure.

[0081] Test Example 2: The surface carboxylated ceramic fluorescent composite particles prepared in step (4) of Example 1 were characterized by Fourier transform red spectroscopy (FT-IR), dynamic light scattering (DLS) technology, and Zeta potential analysis. Figure 7 This is a comparison of the FT-IR spectra of the surface-carboxylated ceramic fluorescent composite particles obtained in step (4) of Example 1 of the present invention and the surface-carboxylated ceramic fluorescent composite particles obtained in step (2). The figure shows that both types of ceramic fluorescent composite particles have a peak value of 1100 cm⁻¹. -1 The vicinity exhibits strong Si-O-Si asymmetric stretching vibration peaks attributed to the silica framework. Compared to uncarboxylated ceramic fluorescent composite particles, carboxylated ceramic fluorescent composite particles show peaks in the 1700–1730 cm⁻¹ range. -1 The appearance of C=O stretching vibration peaks belonging to carboxyl groups proves that the surface of ceramic fluorescent composite particles has been successfully carboxylated.

[0082] Figure 8 The following are the DLS characterization results of the surface carboxylated ceramic fluorescent composite particles in Example 1 of this invention. The results show that the average hydrated particle size of the prepared fluorescent composite particles is about 259.5 nm, and the polydispersity index (PDI) is 0.072, indicating that the carboxylated ceramic fluorescent composite particles have excellent monodispersity and no obvious agglomeration.

[0083] Figure 9The zeta potential characterization results are shown for the surface carboxylated ceramic fluorescent composite particles in Example 1 of this invention. The results show that the average zeta potential of the carboxylated ceramic fluorescent composite particles is -38.9 mV, demonstrating the excellent colloidal stability of the carboxylated ceramic fluorescent composite particles.

[0084] Example 2: Ceramic fluorescent composite particles with high fluorescent nanocrystal loading A ceramic fluorescent composite particle with high fluorescent nanocrystal loading, the specific steps are as follows: (1) Preparation of mesoporous silica: Mesoporous silica was prepared according to Example 1.

[0085] (2) Preparation of silica-dense coated Sr doped CsPbBr3 fluorescent composite particles: Add 0.3 mmol cesium bromide, 0.3 mmol lead bromide, 0.3 mmol strontium bromide, 0.3 mmol potassium carbonate and 100 mL water to a 100 mL beaker, sonicate at room temperature for 5 minutes, then add 0.5 g of the above mesoporous silica, stir at 90 °C until the mixture is dry, and sinter at 400 °C for 3 h to obtain ceramic fluorescent composite particles with high fluorescent nanocrystal loading.

[0086] Figure 10 This is the energy-dispersive X-ray spectrum of the ceramic fluorescent composite particles with high fluorescent nanocrystal loading in Example 2 of the present invention. The figure shows that the characteristic element signal intensity of the Sr-doped CsPbBr3 fluorescent nanocrystals in the ceramic fluorescent composite particles is significant. The mass percentage (Weight %) of the CsPbBr3 fluorescent nanocrystals is 29%, demonstrating the ultra-high loading of fluorescent nanocrystals achieved by the preparation process of the present invention.

[0087] Figure 11 The absolute quantum yield (PLQY) spectrum of the ceramic fluorescent composite particles with high fluorescent nanocrystal loading in Example 2 of this invention is shown. By integrating and comparing the emission peak area with the number of absorbed excitation photons (i.e., the area difference between the blank and the sample at the excitation peak), the absolute PLQY of the sample was calculated to be 98.7%.

[0088] Figure 12 The absolute quantum yield (PLQY) spectra of ceramic fluorescent composite particles with high fluorescent nanocrystal loading prepared according to step (2) of Example 1 of this invention, with the addition of excess cesium bromide and lead bromide, were measured. The absolute PLQY of this sample was significantly reduced to 44.5%.

[0089] Example 3: Preparation of a ceramic fluorescent probe test strip for respiratory syncytial virus (RSV) detection like Figure 13As shown, the respiratory syncytial virus ceramic fluorescent probe test strip described in this embodiment adopts a stacked structure. The test strip includes a PVC base plate as a support layer. On the PVC base plate, along the sample flow direction (from upstream to downstream), the following components are sequentially overlapped: a sample pad, a conjugate pad, a chromatography membrane, and an absorbent pad. Adjacent components maintain a 1-2 mm overlap area to ensure the continuity of liquid chromatography.

[0090] The conjugate pad is loaded with the respiratory syncytial virus ceramic fluorescent probe of Example 1.

[0091] The chromatography membrane is equipped with a detection line and a control line. The detection line is coated with respiratory syncytial virus detection antibody, and the control line is coated with negative control secondary antibody.

[0092] The test strips are prepared using conventional production processes in the field of immunochromatography. For details, please refer to the "Guidelines for Inspection of Quality System of In Vitro Diagnostic Reagent Production (2017 Revised Edition)".

[0093] To verify the sensitivity and detection range of the respiratory syncytial virus ceramic fluorescent probe test strip prepared in this embodiment, the following tests were performed: (1) Sample preparation: Using 50mM Tris-HCl buffer, respiratory syncytial virus antigen (recombinant respiratory syncytial virus protein) was prepared into 0 ng / mL negative control and RSV simulated positive sample.

[0094] (2) Sample addition operation: Take 70 μL of the above-mentioned test samples of different concentrations and add them vertically to the sample wells of different test strips.

[0095] (3) Reaction and reading: Let the reaction stand at room temperature for 15 minutes. After the liquid has completely chromatographically transferred to the absorbent pad, place the test strip under a UV lamp or a fluorescence immunoassay analyzer and observe and record the fluorescence color development of the test line (T line) and the control line (C line).

[0096] Figure 14 The results of the respiratory syncytial virus ceramic fluorescent probe test strip under ultraviolet light excitation are shown after 15 minutes of chromatographic reaction. Figure 14 As shown, the right-hand test strip shows a negative result: a single fluorescent band is observed only at the control line (C line), and no fluorescent signal is observed at the test line (T line), indicating that the sample does not contain the target respiratory syncytial virus (RSV), and the color development of the C line proves that the test strip chromatography process is effective. The left-hand test strip shows a positive result: clear fluorescent bands are observed at both the test line (T line) and the control line (C line) (i.e., double-band phenomenon), indicating that the probe specifically binds to the recombinant RSV protein and accumulates at the T line, confirming the effective detection capability of the test strip for RSV.

[0097] Recombinant respiratory syncytial virus (RSV) protein was diluted in 50 mM Tris-HCl buffer to prepare RSV-simulated positive samples at different concentrations. The fluorescence intensity of the test strips at different RSV protein concentrations was detected using a fluorescence immunoassay analyzer. The results are shown below. Figure 15 As shown, within the respiratory syncytial virus recombinant protein concentration range of 0 to 200 ng / mL, the signal intensity ratio (T / C ratio) of the test line (T line) to the control line (C line) of the test strip exhibits a good linear relationship with the concentration. The regression curve for this linear relationship is Y = 0.0417X + 0.1046 (where Y is the T / C ratio and X is the recombinant protein concentration), and its linear correlation coefficient (R²) is [missing value]. 2 The response rate is 0.997. This highly linear response confirms that the present invention has excellent quantitative detection capability within this detection range, and that the test strip has extremely high detection sensitivity.

[0098] Example 4: Preparation of fluorescent probe for human chorionic gonadotropin A method for preparing a fluorescent probe for human chorionic gonadotropin, the specific steps of which are as follows: (1) Preparation of mesoporous silica / alumina material: Add 1 g CTAB, 0.2 g F127, 480 mL water and 0.2 g sodium hydroxide to a 1000 mL beaker. Stir at 60 °C for 0.5 h, then add 20 mL TEOS and 3 mmol aluminum isopropoxide. Continue heating and stirring for 5.5 h, then stop the reaction. After the solution returns to room temperature, centrifuge and dry the precipitate at 70 °C. Grind the precipitate and add 150 mL isopropanol. Reflux overnight to release the mold.

[0099] (2) Preparation of densely coated CsPbBr3 fluorescent composite particles of silica / alumina: Add 0.5 mmol of cesium bromide, 0.5 mmol of lead bromide and 100 mL of water to a 100 mL beaker, disperse by ultrasonication, and then add 0.35 g of the above mesoporous silica / alumina. Stir and dry at 90 °C and sinter at 900 °C for 4 h to obtain ceramic fluorescent composite particles.

[0100] (3) Preparation of surface carboxyl-functionalized ceramic fluorescent composite particles: The above ceramic fluorescent composite particles were dispersed in 40 mL of ultrapure water and 100 μL of 3-(methacryloyloxy)propyltrimethoxysilane was added. The mixture was sonicated at room temperature for 0.5 h, washed twice with water, and then placed in a 100 mL three-necked flask. The flask was evacuated, nitrogen gas was introduced, and the temperature was heated to 75 °C. 200 μL of acrylic acid and 10 mg of potassium persulfate were injected into the three-necked flask. The mixture was reacted for 24 h to obtain carboxyl-functionalized ceramic fluorescent composite particles.

[0101] (4) Preparation of human chorionic gonadotropin fluorescent probe: 200 μg of the above-mentioned surface carboxyl-functionalized ceramic fluorescent composite particles were dispersed in 500 μL phosphate buffer (50 mM, pH=7.4), 100 μg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 180 μg of N-hydroxysuccinimide sodium salt were added, and the mixture was incubated at room temperature for 30 minutes. Then, 30 μg of mouse anti-human chorionic gonadotropin β subunit monoclonal antibody was added, and the mixture was incubated at 4°C overnight. After washing, the mixture was blocked with 5% bovine serum albumin for 30 minutes and washed again to obtain the human chorionic gonadotropin fluorescent probe.

[0102] Comparative Example 1 The surface modification of silica-coated CsPbBr3 core-shell nanocrystals prepared by the traditional liquid-phase method is carried out through the following steps: (1) Preparation of silica-coated CsPbBr3 core-shell nanocrystals: 0.1468 g PbBr2, 0.0851 g CsBr, 0.6 mL oleylamine (OAm), and 1.8 mL oleic acid (OA) were added to 10 mL N,N-dimethylformamide (DMF). The mixture was stirred at 90 °C for 2 hours to obtain a clear precursor solution. 2 mL of this precursor solution was taken, and 40 µL of 2.8% ammonia solution was added to it for later use. 0.2 mL of the above-mentioned prepared precursor solution was quickly injected into 10 mL of anhydrous toluene containing 5 µL of tetramethyl orthosilicate (TMOS). The system was then vigorously stirred at 1500 rpm. After 10 seconds, the stirring speed was adjusted to 150 rpm, and the reaction was continued for 120 minutes. After the reaction was completed, the final product was collected by centrifugation at 9000 rpm for 5 minutes.

[0103] (2) Carboxyl-functionalized silica-coated CsPbBr3 core-shell nanocrystals were prepared using the same steps as in Example 4 (i.e., step (3) of Example 4).

[0104] Result: As Figure 16 As shown, when the obtained silica-coated CsPbBr3 core-shell nanocrystals were dispersed in an aqueous phase for modification, their photoluminescence intensity rapidly decayed to quench within 40 minutes. This indicates that the silica shell was completely ineffective in preventing moisture from eroding the perovskite core, and the purpose of modification after coating was completely failed. In contrast, the fluorescent probe of Example 1 of this invention did not decay within 40 minutes.

[0105] Example 4: Preparation and specific imaging of a ceramic fluorescent probe targeting the KCNQ1 protein in the HEK 293T cell membrane. (1) Cell culture and transfection: HEK 293T cells (human embryonic kidney cells) were seeded in confocal culture dishes. DMEM containing 10% fetal bovine serum was used for culture at 37 ℃ and 5% CO2. When the cells reached 80% confluence, the KCNQ1 overexpression plasmid was transfected into the cells using Lipofectamine 3000 liposome transfection reagent.

[0106] (2) Probe labeling and incubation a. Probe coupling: Take the surface carboxylated ceramic fluorescent composite particles prepared in step (4) of Example 1, and couple them with anti-KCNQ1 antibody using the EDC / NHS activation method. Remove the free antibody by centrifugation, and redisperse the precipitate in PBS buffer to obtain a fluorescent probe solution coupled with the antibody.

[0107] b. Cell incubation: 24 h after transfection, cells were fixed in PBS with 4% paraformaldehyde for 15 minutes, washed three times with PBST, and blocked with goat serum. The conjugated fluorescent probe solution was added, and the cells were incubated overnight at 4°C, followed by washing three times with PBST. DAPI staining solution was added to counterstain the cell nuclei (blue fluorescence) to aid in localization.

[0108] The labeled cells were imaged and observed using a laser scanning confocal microscope. The specific procedure was as follows: the excitation wavelength was set to 408 nm, and fluorescence signals were acquired under a 40x oil immersion objective.

[0109] The results are as follows Figure 17 As shown. Figure 17 The results showed that the fluorescence signal was mainly distributed in the cell membrane and intracellular region; this indicates that the ceramic fluorescent probe of the present invention can effectively fluorescently label both the cell membrane structure and the cytoplasm at the same time.

[0110] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A ceramic fluorescent composite particle, characterized in that, The ceramic fluorescent composite particles comprise multiple fluorescent nanocrystals and inorganic materials; the inorganic matrix is ​​derived from mesoporous materials, and the multiple fluorescent nanocrystals are dispersed within the pore structure of the mesoporous material; the ceramic fluorescent composite particles are obtained through sintering, during which the framework of the inorganic matrix shrinks and densifies, causing the pore structure to partially or completely close, thereby forming a dense coating layer on the surface of the fluorescent nanocrystals; the density of the ceramic fluorescent composite particles is 1.1 g / cm³. 3 Up to 6 g / cm 3 The fluorescent nanocrystals are loaded into the total mass of the ceramic fluorescent composite particles at a rate of 10 wt% to 35 wt%; the absolute quantum yield of the ceramic fluorescent composite particles is above 85%.

2. The ceramic fluorescent composite particles according to claim 1, characterized in that, The mesoporous material is selected from at least one of mesoporous single oxides or mesoporous composite oxides. The mesoporous single oxide includes at least one of mesoporous silica, mesoporous alumina and mesoporous titanium dioxide; The mesoporous composite oxide includes mesoporous silicon dioxide-metal oxide.

3. The ceramic fluorescent composite particles according to claim 1, characterized in that, The structure of the fluorescent nanocrystals is selected from any of the following: (1) The perovskite structure has a stoichiometric ratio of ABX3; where B is selected from Sn, Pb, Cu, Mn, Ca, Sr, Ba or a combination thereof; (2) Non-perovskite structure, the stoichiometric ratio of which is selected from ABX3, A4BX6 or A2BX5; wherein B is selected from Pb, Zn, Ca, Ba or a combination thereof; In any of the above structures, A is independently selected from Rb, K, or Cs; X is independently selected from F, Cl, Br, or I.

4. The ceramic fluorescent composite particles according to claim 1, characterized in that, The sintering temperature is 400-900℃ and the time is 2.5-5h.

5. The ceramic fluorescent composite particles according to claim 2, characterized in that, The mesoporous silica has a particle size of 20-600 nm and a pore size of 2-15 nm. The fluorescent composite particles with the mesoporous silica as the framework are sintered at a temperature of 400-600℃, and / or... The mesoporous silica-metal composite is mesoporous silica-alumina, and the sintering temperature of the fluorescent composite particles with the mesoporous silica-metal composite as the framework is 600-900℃.

6. The ceramic fluorescent composite particles according to any one of claims 1-3, characterized in that, The fluorescent nanocrystals have a CsPb structure. 1-x Sr x Br3 where 0 < x < 1.

7. The method for preparing ceramic fluorescent composite particles according to any one of claims 1-6, characterized in that, Includes the following steps: S1. Cesium salt and metal salt are added to an aqueous medium and ultrasonically treated at room temperature to form a dispersion, wherein the metal salt is lead salt and / or strontium salt; S2. Add mesoporous silica or mesoporous silica / alumina to the dispersion, stir and evaporate at 80-100℃ until the water evaporates completely to obtain a solid mixture; then sinter the solid mixture at 400-900℃ for 2.5-5h to obtain the ceramic fluorescent composite particles.

8. The use of the ceramic fluorescent composite particles according to any one of claims 1-6, characterized in that, The intended use includes applying the ceramic fluorescent composite particles to: (a1) Prepare fluorescent immunochromatographic test strips or kits; (a2) Prepare fluorescent probes for in vitro cell imaging or in vivo fluorescence imaging; (a3) Prepare drug delivery carriers or drug controlled release systems.

9. A fluorescent probe, characterized in that, The fluorescent probe includes: The core particles comprise the ceramic fluorescent composite particles according to any one of claims 1-6; A surface modification layer that covers the surface of the core particles; And biometric molecules, which are fixed on the surface modification layer; The surface modification layer includes a silane coupling agent-derived layer, and optionally, a polymer coating layer; the silane coupling agent-derived layer and the polymer coating layer provide reactive groups, which are covalently linked to the biorecognition molecule. The reactive group is selected from at least one of carboxyl, thiol, amino, epoxy, aldehyde or maleimide.

10. A method for preparing a fluorescent probe, characterized in that, Includes the following steps: S1: Surface modification treatment is performed on the ceramic fluorescent composite particles according to any one of claims 1-6 to obtain surface-functionalized ceramic fluorescent composite particles; the surface modification treatment includes at least one of the following methods: (a) Silanization-derivation modification: The ceramic fluorescent composite particles are contacted with a silane coupling agent containing an anhydride group and then hydrolyzed; or, the ceramic fluorescent composite particles are first contacted with an amino-containing silane coupling agent and then ring-opening reaction is carried out with an anhydride compound. (b) Silane-polymer coupling modification: First, the ceramic fluorescent composite particles are contacted with an amino-containing silane coupling agent to introduce surface amino groups; then, under the action of an activator, the surface amino groups undergo a condensation reaction with a carboxyl-containing polymer. (c) Surface in-situ polymerization modification: First, the ceramic fluorescent composite particles are contacted with a silane coupling agent containing polymerizable active groups to introduce surface active sites; then, under the action of an initiator, the carboxyl-containing monomers are grafted and polymerized on the surface of the particles. S2. Disperse the surface-functionalized ceramic fluorescent composite particles in a buffer solution with a pH of 5.0-9.5, add a cross-linking agent and protein, and incubate to obtain a fluorescent probe of surface-modified protein.

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