CN1523076A - A magnetic fluorescent bifunctional microsphere with core-shell structure and preparation method thereof - Google Patents

Abstract

The present invention relates to a magnetic fluorescent double-functional microsphere with core-shell structure and its preparation method. Firstly, said invention uses strong acid and sodium citrate to modify surface of inorganic magnetic nano granules, then uses alkyl orthosilicate as precursor body, and adopts gel-sol process to prepare silicon dioxide magnetic microsphere with core-shell structure, finally utilizes the copolycondensation method of alkyl orthosilicate and silane coupling agent of bonded fluorescein to prepare silicon dixide fluorescent microsphere with magnetism. Said microsphere has magnetic response characteristic, and can send out fluorescence under the excitation of UV-light and visible light.

Description

A magnetic fluorescent bifunctional microsphere with core-shell structure and preparation method thereof

technical field

The invention belongs to the technical field of inorganic materials, and in particular relates to a magnetic fluorescent bifunctional microsphere with a core-shell structure and a preparation method thereof.

technical background

Functional microspheres are a class of microspheres with special functions, such as magnetic microspheres, fluorescent microspheres, pH-sensitive microspheres, temperature-sensitive microspheres, and surface-modified microspheres with various Microspheres with functional groups, etc.

Fluorescent microspheres are functional microspheres loaded with fluorescent molecules. In recent years, with the in-depth research on fluorescent probe technology, people have been able to prepare various fluorescent microspheres with particle sizes ranging from nanometer to submicron. ball. Due to the enrichment of fluorescent organic molecules in a single microsphere, fluorescent microspheres have broad application prospects in many fields, such as biomedicine, material science, environmental detection, clinical medicine, gene analysis, and cell imaging.

As far as the preparation methods of fluorescent microspheres are concerned, the current literature reports can be divided into three categories. One is to load organic fluorescent molecules onto the microspheres by physical adsorption (mainly electrostatic interaction); The first way is to load the organic fluorescent molecules on the microspheres; the third is to load the organic fluorescent molecules on the microspheres by chemical bonding. Renault et al. connected fluorescent molecules to the surface of functionalized microspheres by electrostatic adsorption to prepare fluorescent microspheres with fluorescent molecules on the surface (Renault R.M, Denjean P, Pansu R.B, Sensors Actuators, 1999(59): 108) . Campbell et al. used the polar interaction between fluorescent molecules and polymer molecules to physically embed fluorescent molecules to prepare fluorescent microspheres containing fluorescent molecules (Campbell A.I, Bartlett P, J.Colloid Inter.Sci., 2002(256): 325). Jardine et al prepared fluorescent microspheres containing fluorescent molecules by bonding fluorescent molecules to a polymerizable monomer and then copolymerizing them with another monomer (Jardine R.S, Bartlett P, Colloids Surf.A, 2002 (211):127).

Magnetic microspheres have broad application prospects in many fields due to their unique magnetic responsiveness and easy functionalization. In fact, as early as the 1970s, Dutch scientist Ugelstad had prepared monodisperse micron-sized polystyrene magnetic microspheres for the first time, and successfully developed a series of polystyrene-based magnetic microspheres. Polystyrene microspheres developed by Ugelstad and others have been successfully applied in the fields of cell separation and clinical diagnosis. Subsequently, many research groups in the world have joined in the research of the preparation and application of magnetic microspheres. With the deepening of research, many new applications of magnetic microspheres have been or are being developed, and the application fields of magnetic microspheres have changed from the original Separation and diagnosis extension to other fields of bioengineering, medicine, and medicine, such as targeted drug delivery preparations, immunoassays, cell culture media, etc.

As far as the current research status is concerned, the development of magnetic microspheres is mainly in the following directions: one is to prepare magnetic microspheres with smaller sizes, that is, to prepare submicron or even nanoscale magnetic microspheres; the other is to prepare biocompatible magnetic microspheres. And biodegradable magnetic microspheres (especially the preparation of targeted drug delivery system); the third is to further functionalize the magnetic microspheres to expand their application fields.

As far as the prepared magnetic microspheres are concerned, there are two main categories. One is the magnetic microspheres prepared by wrapping inorganic magnetic particles with synthetic polymers or natural polymers. At present, this kind of research is more, and the technology of microencapsulation is a relatively primitive method for preparing magnetic microspheres. This method is simple to operate, but the shape of the prepared microspheres is irregular and the size is mostly on the micron scale or even larger. Another feature is that the particle size distribution is generally very wide, which makes it difficult for the prepared magnetic microspheres to work in a wide range of fields. Dekker suspended the magnetic particles in polyethyleneimine (PEI) solution, filtered and dried to obtain magnetic microspheres coated with PEI (Dekker R F M. Appl. Biotech., 1989, 22: 289). Cuyper et al treated magnetic nanoparticles with phospholipids to prepare magnetic liposome microspheres (Cuyper MD, JonianM.Langmuir, 1990, 7: 647). Monomer polymerization is the most researched preparation method after Ugelstad, which mainly includes suspension polymerization, dispersion polymerization, emulsion polymerization (including soap-free emulsion polymerization, seed polymerization) and the like. Margel et al. prepared magnetic polyacrylaldehyde microspheres with a particle size ranging from 0.03um to 80um in the presence of oil-soluble initiators, suspension stabilizers and inorganic magnetic particles (Margel S, Beitler U. USP 4,783,336, 1988). Daniel et al. used microsuspension polymerization to obtain hydrophobic magnetic polymer microspheres with a particle size range of 0.03um-5um (Daniel J C, SchuppsierJ L. USP 4,358,388.1982). The particle size of microspheres prepared by suspension polymerization is relatively large, and the particle size distribution is wide. Emulsion polymerization is a widely used method for preparing magnetic polymer microspheres. Furusawa etc. deposited magnetic particles on polymer latex particles with functional groups, and then used seed emulsion polymerization to prepare magnetic microspheres with a sandwich structure (Furusawa K, Nagashima K, Anzai C, Colloid Polym.Sci, 1994, 272 :1104). In China, Sun Zonghua et al. used magnetic fluid as the core and adopted improved emulsion polymerization to prepare magnetic polystyrene microspheres with a particle size range of 0.06um-10um (Qiu Guangming, Sun Zonghua, Chemical Reagent, 1993, 15(4): 324). Ugelstad et al. first proposed the in-situ method to prepare monodisperse magnetic polymer microspheres, which is characterized in that porous polystyrene microspheres are prepared by a two-step swelling method, and then iron salts are precipitated with alkali in the pores of polystyrene microspheres. Thereby monodisperse magnetic polystyrene microspheres were prepared (Ugelstad J, Mork PC, Schmid R, et al, Polym.Int, 1993, 30:15.).

The second is to prepare magnetic microspheres by wrapping nano-magnetic particles with inorganic materials, or by depositing inorganic magnetic particles on the surface of inorganic materials by adsorption. Among them, the inorganic material is mainly silica, because silica is a biologically inert material, and this inorganic composite microsphere is easy to surface functionalize, so it can be widely used in the fields of biomedicine, clinical diagnosis, and immunoassay. In China, Zhu Yihua et al. used monodisperse silica microspheres as a template, and used electrostatic self-assembly to prepare silica magnetic microspheres with both core and shell silica and multilayer magnetic nanoparticles in the middle (Zhu Yihua, A Hong, Yang Xiaoling, Chinese patent, publication number: CN1380133A). Wang Kemin et al. synthesized magnetic fluorescent particles respectively encapsulating water-soluble fluorescent substances of magnetic particles by hydrolysis of ethyl orthosilicate in an inverse microemulsion system (Tan Weihong, Wang Kemin, Xiao Dan, Chinese Patent, Mo Kai No. CN 1342515A), Ramesh et al. Magnetic silica magnetic microspheres were prepared by sonochemical deposition of Fe(CO) ₅ (Ramesh S, Prozorov R, and Gedanken A, Chem. Mater., 1997, 9(12:2996)).

Magnetic fluorescent microspheres are dual-functional microspheres that combine magnetic responsiveness and fluorescence emission. This kind of microspheres can not only respond to an external magnetic field, but also emit fluorescence under excitation light, so it can be used in a wider range of applications. field is applied.

In the present invention, we propose a simple and feasible method for preparing magnetic fluorescent bifunctional microspheres with a core-shell structure. The preparation process has good repeatability, the prepared microspheres are regular in shape, and the particle size is easy to control.

Contents of the invention

The object of the present invention is to propose a magnetic fluorescent bifunctional microsphere with a core-shell structure and a preparation method thereof.

The magnetic fluorescent bifunctional microsphere with core-shell structure proposed by the present invention is a core-shell structure with nano-magnetic particles (such as inorganic ferrite, etc.) as the core and silicon dioxide doped with organic fluorescent molecules as the shell. The magnetic microspheres have a particle size ranging from nanometer to submicron, as small as 20 nanometers and as large as 200-400 nanometers. Due to the addition of organic fluorescent molecules during the preparation process, the microspheres not only have magnetic responsivity, but also emit fluorescence under the excitation of ultraviolet and visible light, so we call them magnetic fluorescent microspheres.

The shell layer of the above-mentioned microspheres is silicon dioxide prepared from an alkyl orthosilicate as a precursor, and the organic fluorescent molecules doped therein can be fluorescent molecules with isothiocyanate groups (such as isothiocyanate One or more of ester fluorescein FITC), the silane coupling agent used to bond organic fluorescent molecules is trimethoxyaminopropylsilane, triethoxyaminopropylsilane and other silane coupling agents with amino groups one or more of them.

The above-mentioned magnetic fluorescent microspheres with a core-shell structure proposed by the present invention are prepared by a gel-sol method. First use strong acid (such as nitric acid, hydrochloric acid) and sodium citrate to modify the surface of inorganic magnetic particles, and then use the gel-sol method in alcohol/water system to prepare inorganic magnetic particles with silica as the shell and inorganic magnetic particles as the core. Composite magnetic microspheres. Finally, magnetic silica microspheres are used as seeds to prepare magnetic silica fluorescent microspheres through co-condensation of alkyl orthosilicate and fluorescein-bonded silane coupling agent.

Concrete preparation steps are as follows:

1. Use a strong acid to activate the inorganic magnetic particles: disperse the nano-magnetic particles in deionized water, separate and wash them with a magnet, then disperse them in a 2.0-4.0mol/L strong acid solution, stir for 5-20 minutes; then separate them with a magnet Magnetic particles treated with strong acid and washed 3-5 times with deionized water;

2. Modify the activated inorganic magnetic particles with citrate: add citrate solution to the slurry of inorganic magnetic particles obtained in step (1), stir and disperse, then separate with a magnet, and wash with deionized water for 2- 4 times, finally dispersed in deionized water to obtain a dispersion of modified magnetic particles, the concentration of magnetic particles is 0.5-3.0wt%;

3. Preparation of inorganic composite magnetic microspheres with silica as the shell and inorganic magnetic particles as the core: add the dispersion of the modified magnetic particles to the alcohol/water mixture, add lye and orthosilicic acid under stirring Alkyl ester, keep stirring for 4-12 hours, the whole reaction is carried out at 20°C-40°C; the whole system is calculated by weight, the modified magnetic particles account for 0.3-3.0%, the lye accounts for 0.8-3.0%, and the water accounts for 10% -20%, alcohol accounts for 70-75%, and the rest is alkyl orthosilicate;

4. Preparation of magnetic fluorescent microspheres with a core-shell structure: Add an ethanol solution of a fluorescein-bonded silane coupling agent and an alkyl orthosilicate to the system after the reaction in the above step (3), and avoid Stirring and reacting for 12-48 hours, wherein, the alkyl orthosilicate and the fluorescein-bound silane coupling agent ethanol solution are added in a volume ratio of 1:1-1:4.

The silane coupling agent bonded to fluorescein in the above preparation method can be prepared by the following method:

The fluorescent molecule with isothiocyanate group and the silane coupling agent with amine group are added into absolute ethanol at a molar ratio of 1:9-1:5 and stirred at room temperature in the dark for 24-48 hours.

The magnetic fluorescent microspheres with a core-shell structure prepared by the above method can be separated under the action of a magnet and washed with deionized water. Because the microsphere has an inorganic magnetic inner core, it has magnetic responsiveness, and because organic fluorescent molecules are doped in its shell silica, it can emit fluorescence under the excitation of ultraviolet and visible light.

In the present invention, the magnetic nanoparticles used may be any one of γ- _Fe2O3 or _Fe3O4 or other ferrite particles _.

In the present invention, the strong acid used is one of nitric acid, sulfuric acid, hydrochloric acid and the like.

In the present invention, the citrate used may be one or more of sodium citrate and potassium citrate.

In the present invention, the alkali used is one of ammonia water, potassium hydroxide, sodium hydroxide, tetramethylammonium hydroxide and the like.

In the present invention, the alcohol used is one or more of methanol, ethanol, isopropanol and the like.

In the present invention, the precursor used may be one of methyl orthosilicate or ethyl orthosilicate.

In the present invention, the silane coupling agent used is a silane coupling agent with amino groups, such as (APS) trimethoxyaminopropylsilane, triethoxyaminopropylsilane, N-(β-aminoethyl) -γ-Propyltriethoxysilane and the like.

In the present invention, the organic fluorescent molecules used are fluorescent substances with isothiocyanate groups such as FITC.

The magnetic fluorescent microspheres with a core-shell structure prepared by the present invention can control the particle size from nanometer to micron. By controlling the proportion of alcohol, water and alkali in the response system and the ratio of alkyl orthosilicate as the precursor dosage, silica magnetic fluorescent microspheres with different particle sizes can be prepared. The microspheres are magnetically responsive and can emit fluorescence when excited by ultraviolet and visible light. The method of the invention is simple and the raw materials are easy to obtain. The prepared composite microspheres have a narrow particle size distribution and can be controlled in size.

At present, the research on magnetic microspheres at home and abroad mainly focuses on their magnetic properties. In comparison, the present invention prepares silica microspheres wrapped with inorganic magnetic particles, and dopes organic fluorescent molecules into the The magnetic fluorescent microspheres with core-shell structure were prepared in the silica shell layer. Microspheres prepared by this method have the following characteristics:

(1) The magnetic core has magnetic responsiveness. (2) The particle size of the microspheres can be adjusted, and the particle size is narrowly distributed. (3) The microsphere has a core-shell structure, and the inorganic core is wrapped by a shell layer of silica. (4) The preparation method of the microsphere is simple and the stability is high.

Detailed ways

Example 1: In a 500mL three-necked bottle, add 5.0g of ferric oxide powder through deionized washing, add 200mL of deionized water, stir and disperse at 600rpm, then add 100mL of nitric acid solution (3.0M), Stir at room temperature for 10 minutes. Then separate with a magnet and wash 3-5 times with deionized water. Stir and disperse the iron ferric oxide slurry obtained after washing in 400mL of sodium citrate solution (0.2M), then separate it with a magnet and wash it with deionized water for 3-5 times, and finally the ferric iron tetroxide obtained is dispersed in 200mL In deionized water, a ferrofluid with a solid content of about 2.0 wt% was prepared.

In a 25mL single-neck round bottom flask, dissolve 0.0356g of fluorescein FITC with 10mL of absolute ethanol, then add 0.01829g of triethoxyaminopropylsilane (APS), and stir the reaction for 48 hours in the dark to obtain the bonded Silane coupling agent of fluorescent molecule FITC.

In a 500mL three-necked bottle, add 2.0g of the previously prepared magnetic fluid, and dilute it with 50mL of deionized water and 200% ethanol, then add 4mL of concentrated ammonia water under high-speed stirring (600rpm), and wait until the temperature of the system reaches 40°C Then add 1mL tetraethyl orthosilicate, keep stirring for 4 hours, add 2mL of the silane coupling agent bonded with fluorescent molecule FITC prepared above, and add 1mL tetraethyl orthosilicate at the same time, continue to stir at 40°C for 24 hours in the dark . The particle size of the prepared magnetic fluorescent microspheres is 70nm.

Example 2: In a 500mL three-necked bottle, add 5.0g of ferric oxide powder through deionized washing, add 200mL of deionized water, stir and disperse at 600rpm, then add 100mL of nitric acid solution (3.0M), Stir at room temperature for 5-8 minutes. Then separate with a magnet and wash 3-5 times with deionized water. Stir and disperse the iron ferric oxide slurry obtained after washing in 400mL of sodium citrate solution (0.2M), then separate it with a magnet and wash it with deionized water for 3-5 times, and finally the ferric iron tetroxide obtained is dispersed in 200mL In deionized water, a ferrofluid with a solid content of about 2.0 wt% was prepared.

In a 25mL single-neck round bottom flask, dissolve 0.0356g of fluorescein FITC with 10mL of absolute ethanol, then add 0.01829g of triethoxyaminopropylsilane (APS), and stir the reaction for 48 hours in the dark to obtain the bonded Silane coupling agent of fluorescent molecule FITC.

In a 500mL three-necked bottle, add 2.0g of the previously prepared magnetic fluid, and dilute it with 50mL of deionized water and 200% ethanol, then add 4mL of concentrated ammonia water under high-speed stirring (600rpm), and wait until the temperature of the system reaches 30°C Then add 2mL tetraethyl orthosilicate, keep stirring for 12 hours, then add 2mL of the above-prepared silane coupling agent bonded with fluorescent molecule FITC, and at the same time add 1mL tetraethyl orthosilicate, continue to stir at 30°C for 12 hours in the dark . The particle size of the prepared magnetic fluorescent microspheres is 95nm.

Example 3: In a 500mL three-necked bottle, add 5.0g of ferric oxide powder through deionized washing, add 200mL of deionized water, stir and disperse at 600rpm, then add 100mL of nitric acid solution (3.0M), Stir at room temperature for 10-20 minutes. Then separate with a magnet and wash 3-5 times with deionized water. Stir and disperse the iron ferric oxide slurry obtained after washing in 400mL of sodium citrate solution (0.2M), then separate it with a magnet and wash it with deionized water for 3-5 times, and finally the ferric iron tetroxide obtained is dispersed in 200mL In deionized water, a ferrofluid with a solid content of about 2.0 wt% was prepared.

In a 25mL single-neck round bottom flask, dissolve 0.0356g of fluorescein FITC with 10mL of absolute ethanol, then add 0.01829g of triethoxyaminopropylsilane (APS), and stir the reaction for 48 hours in the dark to obtain the bonded Silane coupling agent of fluorescent molecule FITC.

In a 500mL three-necked bottle, add 2.0g of the previously prepared magnetic fluid, and dilute it with 50mL of deionized water and 200% ethanol, then add 4mL of concentrated ammonia water under high-speed stirring (600rpm), and wait until the temperature of the system reaches 20°C Then add 3mL tetraethyl orthosilicate, keep stirring for 10 hours, then add 2mL of the above-prepared silane coupling agent bonded with fluorescent molecule FITC, and at the same time add 1mL tetraethyl orthosilicate, continue to stir at 40°C in the dark for 48 hours . The particle size of the prepared magnetic fluorescent microspheres is 135nm.

Example 4: In a 500mL three-necked bottle, add 5.0g of ferric oxide powder through deionized washing, add 200mL of deionized water, stir and disperse at 600rpm, then add 100mL of nitric acid solution (3.0M), Stir at room temperature for 5-10 minutes. Then separate with a magnet and wash 3-5 times with deionized water. Stir and disperse the iron ferric oxide slurry obtained after washing in 400mL of sodium citrate solution (0.2M), then separate it with a magnet and wash it with deionized water for 3-5 times, and finally the ferric iron tetroxide obtained is dispersed in 200mL In deionized water, a ferrofluid with a solid content of about 2.0 wt% was prepared.

In a 25mL single-neck round bottom flask, dissolve 0.0356g of fluorescein FITC with 10mL of absolute ethanol, then add 0.01829g of triethoxyaminopropylsilane (APS), and stir the reaction for 48 hours in the dark to obtain the bonded Silane coupling agent of fluorescent molecule FITC.

In a 500mL three-necked bottle, add 2.0g of the previously prepared magnetic fluid, and dilute it with 50mL of deionized water and 200% ethanol, then add 4mL of concentrated ammonia water under high-speed stirring (600rpm), and wait until the temperature of the system reaches 40°C Then add 4 mL tetraethyl orthosilicate, keep stirring for 8 hours, add 2 mL of the silane coupling agent bonded with fluorescent molecule FITC prepared above, and add 1 mL tetraethyl orthosilicate at the same time, continue to stir at 20 ° C for 12 hours in the dark . The particle size of the prepared magnetic fluorescent microspheres is 170nm.

Claims (10)

1, a kind of magnetic fluorescent dual-function microballoon with nucleocapsid structure is characterized in that with the nano magnetic particle being nuclear, is shell with the silicon-dioxide of organic fluorescence molecular dopant, and its particle diameter is little to 20 nanometers, arrive the 200-400 nanometer greatly.

2, a kind of preparation method with magnetic fluorescent dual-function microballoon of nucleocapsid structure, it is characterized in that the inorganic magnetic particle being carried out surface modification with strong acid and Trisodium Citrate, utilizing the preparation of gel-sol method then in alcohol/aqueous systems is shell with silicon-dioxide, the inorganic magnetic particle is the inorganic compounding magnetic microsphere of nuclear, be seed at last with the magnetic silica microballoon, the method for the silane coupling agent copolycondensation by tetraalkyl orthosilicate and bonding fluorescein makes the silica fluorescent microballoon with magnetic.

3, the preparation method with magnetic fluorescent dual-function microballoon of nucleocapsid structure according to claim 2 is characterized in that concrete steps are as follows:

(1) use strong acid that the inorganic magnetic particle is activated: the nano inorganic magnetic-particle is dispersed in the deionized water, utilize the magnet separating, washing, be dispersed in then in the strong acid solution of 2.0-4.0mol/L, stirred 5-20 minute, utilize magnet to isolate magnetic-particle through strong acid treatment again, and with deionized water wash 3-5 time;

(2) with the inorganic magnetic particle after the citric acid hydrochlorate modification activation: in the inorganic magnetic particulate slurry of step 1 gained, add citrate solution, dispersed with stirring, separate with magnet then, and with deionized water wash 2-4 time, be dispersed in the deionized water at last, obtain the dispersion liquid of the magnetic-particle after the modification, magnetic-particle concentration is 0.5-3.0wt%;

(3) preparation is that shell, inorganic magnetic particle are the inorganic compounding magnetic microsphere of nuclear with silicon-dioxide: the dispersion liquid of the magnetic-particle after the modification is joined in alcohol/water mixed liquid, under agitation add alkali lye and tetraalkyl orthosilicate, keep and stirred 4-12 hour, entire reaction is carried out at 20-40 ℃; Whole system is calculated according to weight, and the magnetic-particle after the modification accounts for 0.3-3.0%, and alkali lye accounts for 0.8-3.0%, and water accounts for 10-20%, and alcohol accounts for 70-75%, and all the other are tetraalkyl orthosilicate;

(4) preparation has the magnetic fluorescent dual-function microballoon of nucleocapsid structure: add the ethanolic soln and the tetraalkyl orthosilicate of the silane coupling agent of bonding fluorescein in the reacted system of above-mentioned steps (3), the lucifuge stirring reaction is 24-48 hour under the room temperature; Wherein, the ethanolic soln of the silane coupling agent of tetraalkyl orthosilicate and bonding fluorescein by volume 1: 1-1: 4 add.

4, the preparation method with magnetic fluorescent dual-function microballoon of nucleocapsid structure according to claim 3 is characterized in that described inorganic magnetic particle is γ-Fe ₂O ₃Or Fe ₃O ₄Or other ferrite particle is a kind of, and described strong acid is a kind of of nitric acid, sulfuric acid, hydrochloric acid.

5, the preparation method with magnetic fluorescent dual-function microballoon of nucleocapsid structure according to claim 3 is characterized in that used alkali is ammoniacal liquor or Tetramethylammonium hydroxide.

6, the preparation method with magnetic fluorescent dual-function microballoon of nucleocapsid structure according to claim 3 is characterized in that used Citrate trianion is one or more of Citrate trianions such as Trisodium Citrate, Tripotassium Citrate.

7, the preparation method with magnetic fluorescent dual-function microballoon of nucleocapsid structure according to claim 3 is characterized in that used alcohol is one or more of methyl alcohol, ethanol, propyl alcohol, 2-propyl alcohol.

8, the preparation method with magnetic fluorescent dual-function microballoon of nucleocapsid structure according to claim 3 is characterized in that used silane coupling agent is the silane coupling agent that has amido.

9, the preparation method with magnetic fluorescent dual-function microballoon of nucleocapsid structure according to claim 3 is characterized in that used fluorescence molecule material is the organic fluorescence molecule that has isothiocyanate group.

10, the preparation method with magnetic fluorescent dual-function microballoon of nucleocapsid structure according to claim 3 is characterized in that used tetraalkyl orthosilicate is one or more of methyl silicate, tetraethoxy.