A quantum dot fluorescent microsphere and its preparation method and application

Abstract

The present invention is applicable to the technical field of organic / inorganic composite microsphere synthesis, and provides a quantum dot fluorescent microsphere, including a seed microsphere, a fluorescent microsphere shell, and a fluorescent particle of a silicon oxide-coated quantum dot distributed in the interlayer between the fluorescent microsphere shell and the seed microsphere by means of a covalent bond; the fluorescent particle is one or more types of fluorescent particles with different fluorescence emission wavelengths; at least one quantum dot can also be fixed on the surface of the fluorescent microsphere shell. The present invention obtains a quantum dot fluorescent microsphere with monochromatic or multicolor fluorescence of different fluorescence intensities by controlling the mass volume ratio of the added fluorescent particles and quantum dots and the type and quantity of the fluorescent particles, and has the advantages of high fluorescence intensity and uniform fluorescence distribution. The present invention also spaces different quantum dots inside or outside the microsphere by means of a polymer and a silicon oxide double shell to improve the accuracy of the coded signal.

Description

Technical Field

[0001] The invention belongs to the technical field of organic / inorganic composite microsphere synthesis, and in particular relates to a quantum dot fluorescent microsphere and a preparation method and application thereof. Background Art

[0002] Liquid suspension chip technology is a multiplex detection and analysis platform that has the ability to simultaneously screen and quantify multiple proteins, cytokines, etc. in the same sample, and has high research and application value in the field of disease diagnosis. The core technology of liquid suspension biochips is to use coded microspheres to qualitatively identify targets, and a sufficient number of codes is essential for high-density (multivariate) analysis of liquid chips. In order for fluorescent coded microspheres to be applied to high-throughput multi-index detection systems, the prepared fluorescent microspheres must have high fluorescence intensity, uniform size, easy surface functionalization, good biocompatibility, and no interference from coded signals.

[0003] At present, fluorescent microspheres are mainly prepared using organic fluorescent dyes. The preparation method is to swell the fluorescent dye into a polymer microsphere carrier such as polystyrene, or to mark the organic dye on the surface of various microspheres. However, organic fluorescent dyes themselves have disadvantages such as easy degradation and photobleaching, which makes organic fluorescent dyes far from meeting the needs of biological detection. Compared with other luminescent materials, quantum dots are considered to be ideal fluorescent coding materials because of their advantages such as wide excitation spectrum, narrow emission spectrum, and emission wavelength that can be adjusted by size.

[0004] At present, quantum dot fluorescent microspheres are mainly prepared by swelling quantum dots into polymer microspheres or fixing quantum dots on the surface of microspheres by assembling them layer by layer. The quantum dot fluorescent microspheres prepared by the first method have problems such as uneven coating, insufficient coating, leakage of quantum dots, and large particle size of microspheres, which easily lead to differences in the intensity of fluorescence of the microspheres themselves. Although the second method can prepare fluorescent microspheres with uniform fluorescence intensity, it usually uses aqueous quantum dots to fix them on the surface of microspheres by amide condensation. However, the fluorescence yield of aqueous quantum dots is not high. When using quantum dot fluorescence intensity to design coding signal gradients, it will result in a small number of fluorescent signal codes.

[0005] At the same time, when preparing multicolor microspheres using the above two methods, the two-color or multicolor fluorescent dyes used are simultaneously loaded inside or on the surface of the microspheres, and different quantum dots or fluorescent materials may have fluorescence resonance energy transfer, and there will be certain interference between two or more fluorescent substances, resulting in limited actual coding accuracy. Therefore, the quantum dot fluorescent microspheres obtained using the above two preparation methods may have the problem of insufficient discrimination of coding information, making them unable to be actually used in biological detection. Summary of the invention

[0006] The embodiment of the present invention provides a quantum dot fluorescent microsphere, aiming to solve the problems existing in the prior art.

[0007] The embodiment of the present invention is implemented as follows: a quantum dot fluorescent microsphere, a quantum dot fluorescent coding microsphere with monochromatic or multicolor fluorescence of different fluorescence intensities, comprising a seed microsphere, a fluorescent microsphere shell, and a silicon oxide-coated quantum dot fluorescent particle distributed in a sandwich between the fluorescent microsphere shell and the seed microsphere by a covalent bond. The fluorescent microsphere shell is formed by a free radical polymerization reaction of a comonomer and a functional group modification agent.

[0008] The quantum dots of the present invention can be selected from two or more emission wavelengths, and silicon oxide particles with different fluorescence can be prepared respectively, including but not limited to one or more of CdS, CdSe, CdTe, CdSSe / ZnS, CdSe / ZnS, InP / ZnS, CuInS / ZnS quantum dots.

[0009] The seed microspheres can be magnetic or non-magnetic, and fluorescent or non-fluorescent.

[0010] The fluorescent particles of the silicon oxide-coated quantum dots of the quantum dot fluorescent microspheres of the present invention are distributed in the interlayer between the fluorescent microsphere shell and the seed microsphere by means of covalent bonds, rather than being limited to the surface of the seed microspheres, thereby effectively increasing the number of fluorescent particles that can be accommodated inside the fluorescent microspheres, thereby increasing the number of fluorescent signals that can be encoded. At the same time, due to the protection of the silicon oxide shell, the fluorescence stability of the quantum dots is also guaranteed to a certain extent.

[0011] Furthermore, the thickness of the silicon oxide shell of the fluorescent particle is 0.01 to 0.25 μm, preferably 0.05 to 0.2 μm. If the shell thickness of the silicon oxide shell is relatively small, when two or more fluorescent particles with different emission wavelengths are distributed in the fluorescent microspheres, the fluorescence resonance energy transfer phenomenon between different quantum dots cannot be avoided, resulting in a decrease in the accuracy of the fluorescent coding. If the thickness of the silicon oxide shell is too large, it will affect the emission fluorescence intensity of the quantum dots and the number of fluorescent microspheres that can be encoded. Therefore, it is necessary to control the thickness of the silicon oxide shell on the surface of the fluorescent particles.

[0012] Furthermore, the ratio of the seed microsphere radius to the silica shell thickness is 2 to 50, preferably 10 to 40. When the seed microsphere particle size is 1 μm, the numerical ratio is less than 2, and the required thickness of the fluorescent particles exceeds the upper limit of the allowable range, i.e., 0.25 μm. Similarly, when the numerical ratio is greater than 50, the required thickness of the fluorescent particles exceeds the lower limit of the allowable range, i.e., 0.01 μm. Therefore, it is necessary to control the numerical ratio of the seed microsphere radius to the fluorescent particle shell thickness (silicon oxide shell thickness).

[0013] Furthermore, the fluorescent particles are one or more types of fluorescent particles with different fluorescence emission wavelengths. When the quantum dot types are multiple with different fluorescence emission wavelengths, multiple types of fluorescent particles with different fluorescence emission wavelengths can be prepared. When two or more types of fluorescent particles are selected to participate in the synthesis of fluorescent microspheres, they are mainly used to prepare dual-color and multi-color fluorescent microspheres. During the preparation of quantum dot fluorescent microspheres, the mass volume ratio of the added fluorescent particles and quantum dots and the type and quantity of the fluorescent particles can be controlled, and the quantum dot fluorescent microspheres obtained are quantum dot fluorescent coded microspheres with monochromatic or multi-color fluorescence of different fluorescence intensities.

[0014] Furthermore, a layer of oily quantum dots can be combined on the surface of the fluorescent microsphere shell to further increase the number of codes on the fluorescent microsphere.

[0015] The present invention also provides a method for preparing quantum dot fluorescent microspheres, which directly synthesizes fluorescent microspheres containing quantum dots by free radical polymerization, comprising the following steps:

[0016] (1) coating one or more quantum dots with a silicon source to obtain fluorescent particles of silicon oxide-coated quantum dots;

[0017] (2) adding a reagent containing unsaturated double bonds to modify the surface of the fluorescent particles obtained in step (1) with unsaturated double bonds;

[0018] (3) The surface-modified fluorescent particles obtained in step (2), seed microspheres, initiator, dispersant, comonomer and functional group modification agent are mixed in a solvent to cause a free radical polymerization reaction.

[0019] The preparation method of the fluorescent particles of silicon oxide-coated quantum dots in step (1) is as follows: placing the quantum dots and a silicon source together in an alkaline solution environment, causing the silicon source to undergo a hydrolysis condensation reaction, thereby forming a silicon oxide shell layer on the surface of the quantum dots, and the mass ratio of the quantum dots to the silicon source is in the range of 0.5 to 5 mg / g.

[0020] The unsaturated double bonds in step (2) can be modified by directly adding a reagent containing unsaturated double bonds during the process of preparing the fluorescent particles.

[0021] In step (3), the process of the free radical polymerization reaction is to add seed microspheres to a solvent for emulsification reaction, react for 6 to 12 hours, then add an initiator, and perform an activation reaction at a certain temperature for 1 to 3 hours, then add a mixture of surface-modified fluorescent particles, comonomers, dispersants, functional group modification agents and solvents to the above reaction solution to continue the copolymerization reaction, and after reacting for 12 to 24 hours, fluorescent microspheres containing functional groups are prepared, and the final particle size is 2 to 10 μm, preferably 4 to 6 μm.

[0022] The free radical polymerization process is to directly add all other materials to the emulsified seed microspheres after activation. Under certain conditions, the substances involved in the polymerization can continue to grow on the surface of the seed microspheres to obtain the fluorescent microspheres, and the fluorescent particles can be stably fixed in the interlayer between the seed microspheres and the fluorescent microspheres through their surface modified unsaturated bonds. Among them, the amount of seed microspheres added is 0.5-10% of the comonomer content, the amount of fluorescent particles added is 0.1-20% of the comonomer content, the amount of initiator added is 0.5-5% of the comonomer content, the amount of dispersant added is 0.1-5% of the comonomer content, the amount of functional group modification reagent added is 5-30% of the comonomer content, the mass volume ratio of comonomer to solvent is 0.01-0.1g / mL, the temperature of the free radical polymerization reaction is preferably 40-85°C, and the stirring speed is preferably 50-400rpm.

[0023] Furthermore, the polymer seed microspheres are one of polystyrene, copolymers of polystyrene and polyacrylic acid, copolymers of polystyrene and polymethacrylic acid, and copolymers of polystyrene and divinylbenzene, and have a particle size of 1 to 10 μm, preferably 2 to 5 μm.

[0024] Furthermore, the comonomer includes, but is not limited to, one or a combination of styrene and divinylbenzene monomers commonly used in polymer microspheres.

[0025] Furthermore, the functional groups in the functional group modification reagent include one or more of carboxyl, hydroxyl, amino, epoxy, and chloromethyl.

[0026] Furthermore, the initiator includes but is not limited to one of azobisisovaleronitrile, azobisisobutyronitrile and BPO.

[0027] Furthermore, the solvent includes an organic alcohol or a mixture of an organic alcohol and water, wherein the organic alcohol includes but is not limited to one or more of ethanol, isopropanol, butanol, ethylene glycol, and diethylene glycol.

[0028] Furthermore, when the surface of the fluorescent particles obtained in step (1) is modified with unsaturated double bonds, a silane coupling agent containing unsaturated double bonds is used, including one or more of vinyl trimethoxy silane, vinyl triethoxy silane, vinyl tri-tert-butoxy silane, vinyl triacetoxy silane, and y-methacryloxypropyl trimethoxy silane.

[0029] Furthermore, the silicon source in step (1) includes but is not limited to one or more of ethyl orthosilicate, methyl orthosilicate, and sodium silicate.

[0030] Furthermore, the molar ratio of the reagent containing unsaturated double bonds added in step (2) to the silicon source added in step (1) is 0.5 to 2, preferably 0.8 to 1.2. When the ratio is small, the distribution density of the unsaturated double bonds contained on the surface of the fluorescent particles is low, which reduces the efficiency of the copolymerization reaction with the comonomer or seed microspheres, thereby affecting the number of fluorescent particles fixed in the fluorescent microspheres. When the ratio is high, the reagent containing unsaturated double bonds will self-polymerize to form non-fluorescent particles, which will also affect the number of fluorescent particles fixed in the fluorescent microspheres. Therefore, it is necessary to control the molar ratio of the reagent containing unsaturated double bonds to the silicon source.

[0031] Furthermore, when the functional group in the functional group modification reagent is chloromethyl, the functional group modification reagent containing chloromethyl group includes but is not limited to at least one of 3-chloro-2-methylpropylene and p-chloromethylstyrene, and then a thiol conversion reagent can be added to convert the chloromethyl functional group into a thiol group. The thiol conversion reagent is mainly a thiourea reagent. The surface-modified thiol group fluorescent microspheres prepared in this way can also be combined with a layer of quantum dots on the surface for the preparation of multicolor fluorescent microspheres. In addition to the above steps (1)-(3), the following steps are also included:

[0032] (a) adding a thiol conversion reagent to prepare quantum dot fluorescent microspheres with surface modified thiol groups;

[0033] (b) fixing a layer of quantum dots on the surface of the quantum dot fluorescent microspheres modified with thiol groups obtained in step (a);

[0034] (c) performing silicon oxide surface coating treatment to obtain quantum dot fluorescent microspheres coated with silicon oxide surfaces;

[0035] (d) performing functional group modification on the surface of the quantum dot fluorescent microspheres coated on the silicon oxide surface, wherein the functional groups include but are not limited to one or more of carboxyl, hydroxyl, amino, epoxy, and thiol, preferably carboxyl.

[0036] Furthermore, the mass ratio of the thiol conversion reagent in step (4) to the functional group modification reagent in step (3) is 0.2 to 1.

[0037] Furthermore, in step (5), the quantum dots fixed on the surface of the quantum dot fluorescent microspheres with surface modified thiol groups are oily quantum dots, including but not limited to one or more of CdS, CdSe, CdTe, CdSSe / ZnS, CdSe / ZnS, InP / ZnS, CuInS / ZnS quantum dots, preferably one of CdSe, CdSe / ZnS, CdSSe / ZnS, and the size is preferably 2 to 30 nm. The types of oily quantum dots selected can be different types to obtain quantum dot fluorescent microspheres with multicolor fluorescence.

[0038] A functional group modification reagent with a chloromethyl group is used to participate in the polymerization reaction of the fluorescent microspheres, and then the thiol group can be modified on the surface of the fluorescent microsphere shell by adding a thiol conversion reagent. Then, through the coordination reaction between the metal ions and the thiol group, a layer of oily quantum dots can be combined on the surface of the fluorescent microsphere shell. After a layer of quantum dots is fixed on the surface of the fluorescent microsphere shell, a step of silicon oxide surface coating is also included to obtain quantum dot fluorescent microspheres coated with silicon oxide surface. The silicon oxide surface coating step is: placing the quantum dot fluorescent microspheres with quantum dots fixed on the surface together with the silicon source in an alkaline solution environment, so that the silicon source undergoes a hydrolysis condensation reaction, thereby forming a silicon oxide shell layer on the surface of the quantum dot fluorescent microspheres. Its advantage is that the oily quantum dots can be firmly fixed on the surface of the microspheres and will not detach from the microspheres, thereby ensuring that the self-fluorescence signal of the prepared quantum dot fluorescent microspheres will not change, and the fluorescence yield of the oily quantum dots is generally higher than that of the aqueous phase quantum dots, so that the prepared quantum dot fluorescent microspheres have high fluorescence intensity, high fluorescence intensity stability and high coding number and other properties.

[0039] The embodiment of the present invention also provides the application of the quantum dot fluorescent microspheres as fluorescent coding materials.

[0040] The present invention controls the mass volume ratio of the added fluorescent particles and quantum dots and the type and quantity of the fluorescent particles, and the obtained quantum dot fluorescent microspheres are quantum dot fluorescent coded microspheres with monochromatic or multicolor fluorescence of different fluorescence intensities. Compared with the traditional layer-by-layer assembly method or swelling method using aqueous phase quantum dots, the quantum dot fluorescent microspheres prepared by the present invention have the advantages of high fluorescence intensity of the label and uniform fluorescence distribution, which is very convenient for fluorescence coding and avoids the disadvantage that the fluorescence intensity of the fluorescent coded microspheres prepared by the traditional preparation method cannot meet the requirements. In addition, the present invention also spaces different quantum dots inside or outside the microspheres by means of polymer and silica double shells, effectively suppressing the fluorescence resonance energy transfer phenomenon between different quantum dots, and greatly improving the accuracy of the coded signal. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is a TEM image of quantum dot silicon oxide nanoparticles prepared in Example 1;

[0042] Figure 2 is a SEM image of the quantum dot fluorescent microspheres prepared in Example 2;

[0043] Figure 3 is a fluorescence intensity scatter plot of different fluorescence coded quantum dot fluorescent microspheres prepared in Example 2, wherein A, B, C, D, and E are respectively the fluorescence intensity of different fluorescence coded quantum dot fluorescent microspheres prepared in Example 2. 564nm :λ 662nmFluorescent microspheres prepared from two fluorescent particles with a ratio of 5:1, 5:2, 1:1, 2:5, and 1:5, respectively;

[0044] Figure 4 This is the fluorescence emission graph of the dual-color fluorescence coded quantum dot fluorescent microspheres prepared in Example 3;

[0045] Figure 5 This is the fluorescence emission diagram of the three-color fluorescence coded quantum dot fluorescent microspheres prepared in Example 4. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0047] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0048] The embodiment of the present invention controls the mass volume ratio of the added fluorescent particles and quantum dots and the type and quantity of the fluorescent particles to obtain quantum dot fluorescent microspheres that are single-color or multi-color fluorescent quantum dot encoded microspheres with different fluorescence intensities. At the same time, when preparing multi-color fluorescent microspheres, the embodiment of the present invention uses a polymer and a silicon oxide double shell to space different quantum dots inside or outside the microspheres, effectively suppressing the fluorescence resonance energy transfer phenomenon between different quantum dots and improving the accuracy of the encoded signal.

[0049] Example 1

[0050] Weigh 50 mg of CdSSe / ZnS oily quantum dots (662 nm), evenly disperse them in 200 ml of anhydrous ethanol, place them in a three-necked flask, stir them mechanically at 200 rpm, and react them at room temperature for 15 minutes. Then add 14 g of ammonia water and 37 g of tetraethyl orthosilicate to the reaction system and continue to react for 24 hours, wherein tetraethyl orthosilicate is added in batches. Centrifugal washing with ethanol and cyclohexane successively obtains silicon oxide fluorescent particles, whose silicon oxide shell thickness is about 35 nm, such as Figure 1 shown.

[0051] Weigh 0.1 g of the prepared silicon oxide fluorescent particles, evenly disperse them in 36 ml of anhydrous ethanol, and stir at room temperature for 15 minutes. Then measure 3.2 g of ammonia water and 3.6 g of γ-(methacryloyloxy)propyltrimethoxysilane, drop them into the reaction solution, and continue the reaction for 24 hours. Wash them by centrifugation with ethanol and deionized water successively to obtain silicon oxide fluorescent particles with carbon-carbon double bonds on the surface.

[0052] Weigh 0.2g of polystyrene seed microspheres (particle size is about 3.1μm), ultrasonically disperse them in 25ml of 0.25% SDS aqueous solution, add them to a three-necked flask, keep a constant temperature water bath at 43℃, stir at 140rpm, and emulsify the microspheres for 12h. After the emulsification reaction is completed, increase the stirring speed to 260rpm, add 0.1g of benzoyl peroxide (BPO) and react for 2h, then add 60mg of the fluorescent particles modified with carbon-carbon double bonds prepared above, 5g of styrene, 0.08g of polyvinyl pyrrolidone (PVP) and 1.5g of methacrylic acid, ultrasonically disperse them in 145ml of 0.25% SDS aqueous solution in an ice bath, and add the obtained solution to the above reaction system and continue to react for 24h. Wash with ethanol to obtain monochromatic fluorescent microspheres modified with carboxyl groups on the surface.

[0053] Compared with the prior art, in the synthesis process of this embodiment, the surface of the quantum dots has a layer of silicon oxide shell, which effectively avoids the influence of free radicals on the fluorescence yield of the quantum dots during the polymerization reaction, and the silicon oxide fluorescent particles containing quantum dots are fixed inside by covalent bonds. In this process, there is no need to perform pore treatment on the fluorescent microspheres, avoiding problems such as quantum dot leakage. At the same time, the free radical polymerization method can make the fluorescent particles not only fixed on the surface of the seed microspheres, but distributed in the interlayer between the fluorescent microsphere shell and the seed microspheres. This embodiment can also adjust the number of fluorescent particles by controlling the addition ratio of comonomers and fluorescent particles and controlling the thickness of the interlayer, greatly increasing the number of fluorescent signals that can be encoded.

[0054] Example 2

[0055] Weigh 50 mg of CdSSe / ZnS oily quantum dots (662 nm), evenly disperse them in 200 ml of anhydrous ethanol, place them in a three-necked flask, mechanically stir at 200 rpm, and react at room temperature for 15 minutes. Then add 10 g of ammonia water and 25 g of tetraethyl orthosilicate to the reaction system and continue to react for 24 hours, wherein tetraethyl orthosilicate is added in batches. Centrifugal washing with ethanol and cyclohexane successively obtains the first type of silicon oxide fluorescent particles.

[0056] Weigh 50 mg of CdSSe / ZnS oily quantum dots (564 nm), evenly disperse them in 200 ml of anhydrous ethanol, place them in a three-necked flask, mechanically stir at 200 rpm, and react at room temperature for 15 minutes. Then add 10 g of ammonia water and 25 g of tetraethyl orthosilicate to the reaction system and continue to react for 24 hours, wherein tetraethyl orthosilicate is added in batches. Centrifugal washing with ethanol and cyclohexane successively obtains the second type of silicon oxide fluorescent particles.

[0057] Weigh the two silica fluorescent particles prepared above and activate them separately. The process is to weigh 0.1g of fluorescent particles and evenly disperse them in 36ml of anhydrous ethanol, and stir at room temperature for 15 minutes. Then measure 3.2g of ammonia water and 3.6g of γ-(methacryloyloxy)propyltrimethoxysilane, add them dropwise to the reaction solution, and continue the reaction for 24h. Centrifuge and wash with ethanol and deionized water successively to obtain silica fluorescent microspheres with C=C double bonds modified on the surface.

[0058] Weigh 0.1g of polystyrene seed microspheres (particle size is about 3.1μm), ultrasonically disperse them in 25ml of 0.25% SDS aqueous solution, add them to a three-necked flask, keep a constant temperature water bath at 43°C, stir at a speed of 140rpm, and carry out microsphere emulsification reaction for 12h. After the emulsification reaction is completed, increase the stirring speed to 260rpm, add 0.1g of benzoyl peroxide (BPO) and react for 2h, then add 40mg of the two surface carbon-carbon double bond modified fluorescent microspheres prepared above, 5g of styrene, 0.06g of polyvinyl pyrrolidone (PVP) and 1g of methacrylic acid, ultrasonically disperse them in 145ml of 0.25% SDS aqueous solution in an ice bath, add the obtained solution to the above reaction system and continue to react for 24h. Wash with ethanol to obtain dual-color fluorescent microspheres modified with carboxyl groups on the surface, with a particle size of about 4.6μm, such as Figure 2 shown.

[0059] Five kinds of microspheres with different coding signals were prepared according to different addition ratios of the two kinds of silicon oxide fluorescent particles. The fluorescence intensity of each coded fluorescent microsphere was tested by fluorescence confocal microscopy. Three microspheres were randomly selected. The results are shown in Figure 2. Figure 3 shown.

[0060] Example 3

[0061] Weigh 50 mg of CdSSe / ZnS oily quantum dots (662 nm), evenly disperse them in 200 ml of anhydrous ethanol, place them in a three-necked flask, mechanically stir at 200 rpm, and react at room temperature for 15 minutes. Then add 14 g of ammonia water and 37 g of tetraethyl orthosilicate to the reaction system and continue to react for 24 hours, wherein tetraethyl orthosilicate is added in batches. Centrifugal washing with ethanol and cyclohexane successively obtains silicon oxide fluorescent particles.

[0062] Weigh 0.1 g of the prepared silicon oxide fluorescent particles, evenly disperse them in 36 ml of anhydrous ethanol, and stir at room temperature for 15 minutes. Then measure 3.2 g of ammonia water and 3.6 g of γ-(methacryloyloxy)propyltrimethoxysilane, drop them into the reaction solution, and continue the reaction for 24 hours. Wash them by centrifugation with ethanol and deionized water successively to obtain silicon oxide fluorescent particles with carbon-carbon double bonds on the surface.

[0063] Weigh 0.2g of polystyrene seed microspheres (particle size is about 3.1μm), ultrasonically disperse them in 25ml of 0.25% SDS aqueous solution, add them to a three-necked flask, keep a constant temperature water bath at 43°C, stir at 140rpm, and emulsify the microspheres for 12h. After the emulsification reaction is completed, increase the stirring speed to 260rpm, add 0.1g of benzoyl peroxide (BPO) and react for 2h, then add 50mg of the surface double bond silane-modified fluorescent particles prepared above, 5g of styrene, 0.08g of polyvinyl pyrrolidone (PVP) and 1.5g of p-chloromethylstyrene, ultrasonically disperse them in 145ml of 0.25% SDS aqueous solution in an ice bath, and add the resulting solution to the above reaction system and continue to react for 24h. The fluorescent microspheres with chloromethyl groups fixed on the surface obtained after washing with ethanol were mixed with an ethanol solution containing 0.5 g of thiourea, reacted for 2 hours, and finally washed with 50% sodium hydroxide solution to obtain monochromatic fluorescent microspheres with thiol groups modified on the surface.

[0064] Weigh 0.1 g of the above-mentioned fluorescent microspheres modified with thiol groups on the surface, add 50 mg of CdSe / ZnS (478 nm) quantum dots, and ultrasonicate for 5 min to obtain a homogeneous transparent solution. Wash with cyclohexane centrifugation 1-2 times to remove free CdSe / ZnS quantum dots, and obtain dual-color fluorescent microspheres.

[0065] Weigh 40 mg of the above-mentioned dual-color fluorescent microspheres and 6 g of polyvinyl pyrrolidone (PVP), disperse them in 200 mL of ethanol solution, add them to a three-necked flask, stir them mechanically at 200 rpm, and react at room temperature for 12 h. Wash them twice by centrifugation with ethanol to obtain a microsphere precipitate, then disperse the precipitate in 200 mL of ethanol, add 1.5 g of ammonia water, stir and react at room temperature for 5 min, the stirring speed is 200 rpm, then slowly add 1.85 g of tetraethyl orthosilicate and continue stirring and reacting for 12 h, and finally wash them by centrifugation with ethanol 3 times to obtain dual-color fluorescent microspheres with silicon oxide coated on the surface.

[0066] Weigh 0.02 g of the above-prepared dual-color fluorescent microspheres coated with silicon oxide, evenly disperse them in 30 mL of deionized water, and stir at room temperature for 15 minutes. Then measure 1 g of ammonia water and 1 g of silane-polyethylene glycol-carboxyl reagent, add them dropwise to the reaction solution, stir mechanically at 200 rpm, and react at room temperature for 24 hours. First, centrifuge and wash with deionized water to obtain dual-color fluorescent microspheres with silicon oxide shells modified with carboxyl groups.

[0067] Compared with the prior art, this embodiment uses a functional group modification reagent with a chloromethyl group to participate in the polymerization reaction of the fluorescent microspheres, and then the thiol group can be modified on the surface of the fluorescent microspheres by adding a thiol conversion reagent. Subsequently, a layer of oily quantum dots can be combined on the surface of the fluorescent microspheres through the coordination reaction between the metal ions and the thiol groups. The advantage is that the oily quantum dots can be firmly fixed on the surface of the microspheres and will not detach from the microspheres, thereby ensuring that the self-fluorescence signal of the prepared quantum dot fluorescent microspheres will not change, and the fluorescence yield of the oily quantum dots is generally higher than that of the water phase quantum dots, so that the prepared quantum dot fluorescent microspheres have high fluorescence intensity and high fluorescence intensity stability.

[0068] Example 4

[0069] Weigh 50 mg of CdSSe / ZnS oily quantum dots (662 nm), evenly disperse them in 200 ml of anhydrous ethanol, place them in a three-necked flask, mechanically stir at 200 rpm, and react at room temperature for 15 minutes. Then add 10 g of ammonia water and 25 g of tetraethyl orthosilicate to the reaction system and continue to react for 24 hours, wherein tetraethyl orthosilicate is added in batches. Centrifugal washing with ethanol and cyclohexane successively obtains the first type of silicon oxide fluorescent particles.

[0070] Weigh 50 mg of CdSSe / ZnS oily quantum dots (564 nm), evenly disperse them in 200 ml of anhydrous ethanol, place them in a three-necked flask, mechanically stir at 200 rpm, and react at room temperature for 15 minutes. Then add 10 g of ammonia water and 25 g of tetraethyl orthosilicate to the reaction system and continue to react for 24 hours, wherein tetraethyl orthosilicate is added in batches. Centrifugal washing with ethanol and cyclohexane successively obtains the second type of silicon oxide fluorescent particles.

[0071] Weigh the two silica fluorescent particles prepared above and activate them separately. The process is to weigh 0.1g of fluorescent particles and evenly disperse them in 36ml of anhydrous ethanol, and stir at room temperature for 15 minutes. Then measure 3.2g of ammonia water and 3.6g of γ-(methacryloyloxy)propyltrimethoxysilane, add them dropwise to the reaction solution, and continue the reaction for 24h. Centrifuge and wash with ethanol and deionized water successively to obtain silica fluorescent microspheres with C=C double bonds modified on the surface.

[0072] Weigh 0.1g of polystyrene seed microspheres (particle size is about 4.0μm), ultrasonically disperse them in 25ml of 0.25% SDS aqueous solution, add them to a three-necked flask, keep a constant temperature water bath at 43°C, stir at 140rpm, and emulsify the microspheres for 12h. After the emulsification reaction is completed, increase the stirring speed to 260rpm, add 0.1g of benzoyl peroxide (BPO) and react for 2h, then add 40mg of the two surface carbon-carbon double bond-modified fluorescent microspheres prepared above, 5g of styrene, 0.06g of polyvinyl pyrrolidone (PVP) and 1g of p-chloromethylstyrene, ultrasonically disperse them in 145ml of 0.25% SDS aqueous solution in an ice bath, and add the resulting solution to the above reaction system and continue to react for 24h. The fluorescent microspheres with chloromethyl groups fixed on the surface obtained after washing with ethanol were mixed with an ethanol solution containing 0.5 g of thiourea, reacted for 2 hours, and finally washed with 50% sodium hydroxide solution to obtain dual-color fluorescent microspheres with thiol groups modified on the surface.

[0073] Weigh 0.1 g of the above-mentioned two-color fluorescent microspheres modified with thiol groups on the surface, add 40 mg of CdSe / ZnS (478 nm) quantum dots, and ultrasonicate for 5 min to obtain a homogeneous transparent solution. Wash with cyclohexane centrifugation 1 to 2 times to remove free CdSe / ZnS quantum dots, and obtain three-color fluorescent microspheres.

[0074] Weigh 40 mg of the above three-color fluorescent microspheres and 6 g of polyvinyl pyrrolidone (PVP), disperse them in 200 mL of ethanol solution, add them to a three-necked flask, stir them mechanically at 200 rpm, and react at room temperature for 12 h. Wash the microspheres twice by centrifugation with ethanol to obtain a microsphere precipitate, then disperse the precipitate in 200 mL of ethanol, add 1.85 g of ammonia water, stir and react at room temperature for 5 min at a stirring speed of 200 rpm, then slowly add 1.85 g of tetraethyl orthosilicate and continue stirring and reacting for 12 h, and finally wash them by centrifugation with ethanol 3 times to obtain three-color fluorescent microspheres with silicon oxide coated on the surface.

[0075] Weigh 0.02 g of the above-prepared three-color fluorescent microspheres coated with silicon oxide, evenly disperse them in 30 mL of deionized water, and stir at room temperature for 15 minutes. Then measure 1 g of ammonia water and 1 g of silane-polyethylene glycol-carboxyl reagent, add them dropwise to the reaction solution, stir mechanically at 200 rpm, and react at room temperature for 24 hours. First, centrifuge and wash with deionized water to obtain the three-color fluorescent microspheres of silicon oxide modified with carboxyl groups.

[0076] 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 and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A quantum dot fluorescent microsphere, It is characterized in that It includes seed microspheres, fluorescent microsphere shells, and fluorescent particles of silicon oxide-coated quantum dots distributed in the interlayer between the fluorescent microsphere shells and the seed microspheres by means of covalent bonds; The method for preparing the quantum dot fluorescent microspheres comprises the following steps: (1) coating one or more quantum dots with a silicon source to obtain fluorescent particles of silicon oxide-coated quantum dots; (2) adding a reagent containing unsaturated double bonds to modify the surface of the fluorescent particles obtained in step (1) with unsaturated double bonds; (3) mixing the surface-modified fluorescent particles obtained in step (2), seed microspheres, an initiator, a dispersant, a comonomer, and a functional group modification agent in a solvent to cause a free radical polymerization reaction; The functional group in the functional group modifying reagent is chloromethyl, and the steps are also included: (a) adding a thiol conversion reagent to prepare quantum dot fluorescent microspheres with surface modified thiol groups; (b) fixing a layer of oily quantum dots on the surface of the quantum dot fluorescent microspheres modified with thiol groups obtained in step (a); (c) performing silicon oxide surface coating treatment to obtain silicon oxide surface coated quantum dot fluorescent microspheres; (d) Functional group modification on the surface of quantum dot fluorescent microspheres coated with silicon oxide.

2. The quantum dot fluorescent microspheres according to claim 1, It is characterized in that The thickness of the silicon oxide shell in the fluorescent particles is 0.01-0.25 μm; and / or the ratio of the radius of the seed microsphere to the thickness of the silicon oxide shell is 2-50.

3. The quantum dot fluorescent microspheres according to claim 1, It is characterized in that The fluorescent particles are one or more types of fluorescent particles having different fluorescent emission wavelengths.

4. The quantum dot fluorescent microspheres according to claim 1, It is characterized in that At least one quantum dot is fixed on the surface of the outer shell of the fluorescent microsphere.

5. The quantum dot fluorescent microsphere according to claim 1, It is characterized in that The seed microspheres are one of polystyrene, a copolymer of polystyrene and polyacrylic acid, a copolymer of polystyrene and polymethacrylic acid, and a copolymer of polystyrene and divinylbenzene; and / or the comonomer includes one or more of styrene and divinylbenzene monomers; and / or the functional groups in the functional group modification reagent include one or more of carboxyl, hydroxyl, amino, epoxy, and chloromethyl; and / or the reagent containing unsaturated double bonds includes one or more of vinyltrimethoxysilane, vinyltriethoxysilane, vinyltri-tert-butoxysilane, vinyltriacetoxysilane, and y-methacryloxypropyltrimethoxysilane.

6. The quantum dot fluorescent microsphere according to claim 1, It is characterized in that The molar ratio of the reagent containing unsaturated double bonds added in step (2) to the silicon source added in step (1) is 0.5-2.

7. The quantum dot fluorescent microsphere according to claim 1, It is characterized in that The oily quantum dots in step (b) include one or more of CdS, CdSe, CdTe, CdSSe / ZnS, CdSe / ZnS, InP / ZnS, and CuInS / ZnS quantum dots.

8. Use of the quantum dot fluorescent microspheres according to any one of claims 1 to 4, It is characterized in that As fluorescent coding material.

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