CN101177607B - Copolymerization type cross-linking polymer fluorescent microspheres and preparation method thereof - Google Patents

Abstract

The invention discloses a fluorescent microsphere of a copolymerized cross-linked polymer. The fluorescent substance in the microsphere is a metal complex, and the metal complex is connected with the cross-linked polymer skeleton through a covalent bond. The invention also discloses the The preparation method of the copolymerization type cross-linked polymer fluorescent microsphere is obtained by free radical copolymerization reaction of the metal complex fluorescent monomer and other multifunctional methacrylate monomers in a mixed solvent under the action of an initiator. The present invention has found a suitable fluorescent substance with high fluorescence efficiency and stability, which is easy to obtain, thereby obtaining high-performance fluorescent microspheres; at the same time, it has found an accurate and suitable method to introduce polymerizable groups on fluorescent molecules, which solves the existing problems. The technical problem fills the technical gap in the field, and provides a new technical basis and development direction for the application of fluorescent microspheres in the fields of biology and medicine.

Description

Copolymerized cross-linked polymer fluorescent microspheres and preparation method thereof

technical field

The invention relates to the technical field of luminescent materials, in particular to a copolymerized cross-linked polymer fluorescent microsphere and a preparation method thereof.

technical background

Fluorescent microspheres, as a new type of carrier material, have very important applications in the fields of biology and medicine, especially the development of proximity scintillation analysis technology based on fluorescent microspheres in recent years and the combination of fluorescent microspheres and flow cytometry. Medical research has provided a new and important technology platform. The application of these technologies can simultaneously perform quantitative determination of multiple components while avoiding complex and time-consuming separation processes. Compared with other traditional methods, it is more micro, automated, and sensitive. Advantages, it not only provides a large-scale, high-efficiency, and high-sensitivity new high-throughput drug screening method, but also creates a new method for gene expression research and medical diagnosis by fluorescent microspheres-flow cytometry. The development of new drugs provides new and powerful means.

Fluorescent microspheres can be divided into two categories: inorganic microspheres and polymer microspheres according to their carriers. In comparison, polymer microspheres can be designed, synthesized and prepared at the molecular level by selecting polymer monomers and polymerization methods. Microspheres The size and particle size distribution of the microspheres are easier to control, and it is easy to introduce various functional groups such as amino groups, hydroxyl groups, carboxyl groups, and aldehyde groups on the microspheres through the selection of functional comonomers to endow the microspheres with specific functions. The designability of this microstructure and function makes polymer microspheres have certain advantages over inorganic microspheres.

At present, most of the fluorescent substances used in fluorescent microspheres are organic fluorescent dyes. Organic fluorescent dyes generally have low fluorescence quantum efficiency, poor fluorescence stability, and prone to photobleaching. Sometimes due to insufficient fluorescence intensity, some complex technical means are required. to improve the signal-to-noise ratio. In recent years, the application of semiconductor nanocrystals in fluorescent microspheres has attracted people's attention due to their high fluorescence quantum efficiency, light photobleaching and long half-life. But neither the synthesis of semiconductor nanocrystals nor their introduction into microspheres is easy. Therefore, it is one of the keys to obtain high-performance fluorescent microspheres to seek suitable fluorescent substances with high fluorescence efficiency and high stability.

The introduction methods of phosphors in microspheres mainly include: (a) physical adsorption, through physical adsorption, fluorescent molecules are adsorbed on the surface of microspheres or wrapped in microspheres. This method is simple and easy, but the combination of fluorescent molecules is not firm. , easy to be lost, especially when used in some non-aqueous environments, organic dyes are easily leached; (b) chemical modification of the microsphere surface, fluorescent molecules form covalent bonds with functional groups on the surface of the microspheres, the shortcomings of this method Fluorescent molecules may occupy too many active sites, making it difficult for bioactive molecules to bind to the surface of microspheres. In addition, fluorescent molecules are exposed to the outside and are easily affected by the external environment and media, making the detection accuracy and reproducibility poor. (c) Fluorescent monomers are copolymerized with other monomers, polymerizable groups are introduced on the fluorescent molecules, and then copolymerized with other monomers. This method combines the fluorescent molecules firmly in the microspheres. The difficulty is to find a suitable method in A polymerizable group is introduced into the fluorescent molecule.

In summary, seeking suitable fluorescent substances with high fluorescence efficiency and stability is one of the keys to obtain high-performance fluorescent microspheres, and finding a suitable method to introduce polymerizable groups on fluorescent molecules is another. key issues to be resolved.

Contents of the invention

An object of the present invention is to provide a fluorescent microsphere of a copolymerized cross-linked polymer, which is selected from metal 8-hydroxyquinoline with high fluorescence efficiency and good light and thermal stability, which has important applications in the field of electroluminescent materials. The complex is a fluorescent substance, and the free radical copolymerization reaction is carried out between the fluorescent monomer containing the complex and the cross-linked acrylate monomer to obtain a copolymerized cross-linked polymer fluorescent microsphere.

Another object of the present invention is to provide a simple and feasible preparation method of the copolymerized cross-linked polymer fluorescent microspheres.

The technical solution of the present invention is to provide a fluorescent microsphere of a copolymerized cross-linked polymer, the fluorescent substance in the microsphere is a metal complex, and the metal complex is connected to the cross-linked polymer skeleton through a covalent bond.

The invention provides a method for preparing the fluorescent microspheres of copolymerized cross-linked polymers, which are obtained by free-radical copolymerization of monomers in a mixed solvent under the action of an initiator, and the monomers include metal complex fluorescent microspheres. monomers and other multifunctional methacrylate monomers.

The mixed solvent is a mixture of tetrahydrofuran and absolute ethanol, wherein the volume content of absolute ethanol in the mixed solvent is 0-20%.

The initiator is azobisisobutyronitrile, and its dosage is 1% of the total mass of monomers.

A dispersant may be added in the copolymerization reaction.

The dispersant is polyvinylpyrrolidone, and its consumption is 20% of the total mass of monomers.

The metal complex fluorescent monomer is the aluminum complex Al(maq) of methacrylic acid-[2-(8-hydroxyquinoline-5)methoxy]-ethyl ester (maq) ₃ or the zinc complex Zn( maq) ₂ , its molecular structure formula is as follows:

The metal complex fluorescent monomer accounts for 1% to 4% of the total monomer mass; the cross-linked acrylate monomer is hexamethylene dimethacrylate (HDMA) and/or ethylene glycol dimethacrylate (EDMA), the dosage accounts for 96% to 99% of the total mass of monomers.

The temperature of the copolymerization reaction is 65°C.

The fluorescent substance in the microsphere is composed of the aluminum complex Al(maq) ₃ or the zinc complex Zn(maq) of the fluorescent monomer methacrylic acid-[2-(8-hydroxyquinoline-5)methoxy]-ethyl ester ₂ is introduced through a copolymerization reaction, so the fluorescent substances it contains are connected to the polymer backbone through covalent bonds. The fluorescent microspheres are prepared by free radical copolymerization of fluorescent monomer Al(maq) ₃ or Zn(maq) ₂ and cross-linked acrylate monomer. In the preferred preparation scheme of the present invention, the fluorescent monomer accounts for 1% to 4% of the total mass of the monomer; the crosslinked acrylate monomer is hexamethylene dimethacrylate (HDMA) or dimethyl Ethylene glycol acrylate (EDMA) or a mixture of the two accounts for 96% to 99% of the total mass of the monomers; the mixed solvent used is a mixture of tetrahydrofuran and absolute ethanol, wherein the volume content of absolute ethanol in the mixed solvent is 0～20%; Used dispersant is polyvinylpyrrolidone (MW 58000), and its consumption is 20% of monomer gross mass; Common oil-soluble free radical polymerization initiator all can be used as polymerization initiator, and the preferred of the present invention is The amount of azobisisobutyronitrile (AIBN) is 1% of the total mass of monomers; the polymerization reaction temperature is 65°C.

The beneficial effect of the present invention is to find a suitable fluorescent substance with high fluorescent efficiency and stability that is easy to obtain, thereby obtaining high-performance fluorescent microspheres; at the same time, an accurate and suitable method is found to introduce polymerizable groups on fluorescent molecules, It solves the problems of the existing technology, fills the technical gap in the field, and provides a new technical basis and development direction for the application of fluorescent microspheres in the fields of biology and medicine.

Description of drawings

Figure 1 Fluorescence spectra of representative fluorescent microspheres

Detailed ways

The present invention will be described in further detail below in conjunction with the accompanying drawings and specific embodiments.

According to the following experimental method, the particle size of the obtained polymer fluorescent microspheres is adjusted by changing the experimental conditions such as the composition of the crosslinking monomer, the composition of the reaction solvent, and the addition of a dispersant. The experimental conditions and results of Examples 1-14 are provided in the table 1 in.

Experimental method: Add 20mL of reaction solvent to a 50ml double-neck round bottom flask, add fluorescent monomer and crosslinking monomer, stir and dissolve evenly, and then bubble _N2 gas for 15 minutes to fully remove O in the reaction solution. ₂ . The initiator AIBN was added under the protection of N ₂ gas, the temperature of the system was raised to 65°C, and the polymerization reaction was carried out under the protection of N ₂ gas for 24 hours.

After the reaction is over, pour the reaction mixture into methanol to precipitate, and then carry out centrifugation. The obtained fluorescent microspheres are washed with methanol for many times, centrifuged, and then washed with distilled water for several times, and vacuum-dried to obtain fluorescent microsphere powder, which can also be directly Store it dispersed in water.

Measurement of particle size and particle size distribution of fluorescent microspheres: the fluorescent microspheres were dispersed in water and measured by a laser particle size analyzer (MasterSizer 2000).

Measurement of the fluorescence spectrum of the fluorescent microspheres: the fluorescent microspheres were made into a 5×10 ^-5 g/ml aqueous suspension, and measured on a Shimadzu RF-5301PC fluorescence spectrophotometer, with an excitation wavelength of 393nm.

Table 1 The experimental results of fluorescent microsphere preparation under different conditions

Example Fluorescent monomer*(g) Cross-linking monomer composition (g) Solvent composition (mL) D[4,3](μm) consistency HDMA EDMA THF EtOH 1 0.02 0 0.98 20 0 14.845 0.43772

2 0.02 0.24 0.74 20 0 19.014 0.649132 3 0.02 0.49 0.49 20 0 21.602 0.63829 4 0.02 0.74 0.24 20 0 11.756 0.445607 5 0.02 0.98 0 20 0 9.42 0.509454 6 0.02 0.98 0 19 1 7.097 0.480506 7 0.02 0.98 0 18 2 5.421 0.406605 8 0.02 0.98 0 17 3 10.843 0.418734 9 0.02 0.98 0 16 4 10.103 0.582782 10 0.01 0.99 0 18 2 12.311 0.456225 11 0.04 0.96 0 18 2 10.466 0.406426 12 0.02 0.98 0 18 2 12.551 0.493648 13** 0.02 0.98 0 20 0 8.608 0.4094 14** 0.02 0.98 0 19 1 4.566 0.3729

Initiator: AIBN, 0.01g; Polymerization temperature: 65°C;

*The fluorescent monomer of Example 12 is Zn(maq) ₂ ; Except this embodiment, the fluorescent monomer of other embodiments is Al(maq) ₃ ;

**Add 0.2g polyvinylpyrrolidone dispersant

Accompanying drawing 1 is the fluorescent spectrum of representative fluorescent microsphere, and the strongest emission wavelength of gained fluorescent microsphere when fluorescent monomer is Al (maq) ₃ and Zn (maq) ₂ is respectively 510nm and 520nm, by embodiment 7,10 Compared with 11, it can be seen that with the increase of the content of fluorescent monomer, the fluorescence intensity of microspheres increases, and the content of fluorescent monomer can be adjusted according to actual needs.

Claims (7)

1. A copolymerization type cross-linked polymer fluorescent microsphere is characterized in that said copolymerization type cross-linked polymer fluorescent microsphere is made of a monomer under the action of an initiator, in a solvent, in the presence of a dispersant through free radicals Obtained by a copolymerization reaction, the fluorescent substance in the microsphere is a metal complex; the monomer includes a metal complex fluorescent monomer and other multifunctional methacrylate monomers; the other multifunctional methacrylate monomers Hexamethylene dimethacrylate and/or ethylene glycol dimethacrylate; the metal complex fluorescent monomer is methacrylic acid-[2-(8-hydroxyquinoline-5)methoxyl]-ethyl Aluminum complex Al(maq) ₃ or zinc complex Zn(maq) ₂ of the ester, the complex is connected to the cross-linked polymer skeleton through covalent bonds; the solvent is tetrahydrofuran or a mixture of tetrahydrofuran and absolute ethanol , wherein the volume content of absolute ethanol in the solvent is 0-20%. 2. according to the described copolymerization type cross-linked polymer fluorescent microsphere of claim 1, it is characterized in that the molecular structural formula of described metal complex fluorescent monomer is as follows respectively:

3. a preparation method of the described copolymerization type cross-linked polymer fluorescent microsphere of claim 1, it is characterized in that described cross-linked polymer fluorescent microsphere is made of metal complex fluorescent monomer and other multifunctional methacrylate The monomer is obtained by free radical copolymerization under the action of an initiator, in a solvent, and in the presence of a dispersant; the solvent is tetrahydrofuran or a mixture of tetrahydrofuran and absolute ethanol, wherein the volume content of absolute ethanol in the solvent is 0 ~20%. 4. according to the preparation method of the described copolymerization type cross-linked polymer fluorescent microsphere of claim 3, it is characterized in that described initiator is azobisisobutyronitrile, and its consumption is 1% of monomer gross mass. 5. The method for preparing fluorescent microspheres of copolymerized cross-linked polymers according to claim 3, characterized in that the dispersant is polyvinylpyrrolidone, and its consumption is 20% of the total mass of the monomers. 6. according to the preparation method of the described copolymerization type cross-linked polymer fluorescent microsphere of claim 3, it is characterized in that the fluorescent monomer of the metal complex accounts for 1%～4% of the total mass of the monomer; the other multi-functional The amount of the methacrylate monomer accounts for 96%-99% of the total mass of the monomers. 7. The preparation method of the copolymerized cross-linked polymer fluorescent microspheres according to claim 3, characterized in that the polymerization reaction temperature is 65°C.

Images