CN110484234B - Fluorescent microsphere and preparation and fluorescent coding method thereof - Google Patents

Fluorescent microsphere and preparation and fluorescent coding method thereof Download PDF

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CN110484234B
CN110484234B CN201810458170.2A CN201810458170A CN110484234B CN 110484234 B CN110484234 B CN 110484234B CN 201810458170 A CN201810458170 A CN 201810458170A CN 110484234 B CN110484234 B CN 110484234B
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CN110484234A (en
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褚先锋
李为公
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Shenzhen Weigong Biotechnology Co ltd
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Abstract

A fluorescent microsphere and a preparation method and a fluorescent coding method thereof comprise the following steps: mixing the microsphere with a silane reagent and a solvent for activation to obtain an activated microsphere; wherein the silane reagent is at least one of epoxy silane reagent, chloro silane reagent, isocyanic acid silane reagent, succinic anhydride silane reagent and chlorosulfonyl phenyl silane reagent; and (3) carrying out coupling reaction on the activated microsphere and the fluorescent dye in a solvent to obtain the fluorescent microsphere. The preparation method is to couple one or more fluorescent dyes on the surface of the activated microsphere to obtain one or more different fluorescent signals. And simultaneously, the mass volume ratio of each fluorescent dye is regulated, so that one or more microspheres with different fluorescence and different intensities can be obtained, and the fluorescent coding microsphere is obtained. The prepared fluorescent microsphere has the advantages of large fluorescent dye coding space, high fluorescent dye strength and the like, and the defect that the fluorescent intensity of the fluorescent coding microsphere prepared by the traditional preparation method cannot meet the requirement is avoided.

Description

Fluorescent microsphere and preparation and fluorescent coding method thereof
Technical Field
The invention relates to the technical field of biological molecular markers, in particular to a fluorescent microsphere and a preparation method and a fluorescent coding method thereof.
Background
The suspension microarray technology based on fluorescent microspheres and fluorescent codes thereof has the capability of simultaneously screening and quantifying various proteins, cytokines and the like in the same sample, and has high research and application values in the field of disease diagnosis. The core technology of the suspension microarray technology is fluorescent coding microspheres with unique marking signals in the detection process, and in order to enable the fluorescent coding microspheres to be applied to a high-flux multi-index detection system, the fluorescent coding microspheres are required to have the characteristics of high fluorescence intensity, uniform size, easy surface functionalization, good biocompatibility and the like.
The traditional preparation method of fluorescent coding microsphere mainly uses a swelling method, and the preparation method is that fluorescent dye is swelled into a macromolecular microsphere carrier such as polystyrene and the like. In addition, there is a method of preparing a silanized fluorescent dye coated on the surface of the microsphere. However, the fluorescent dye prepared by the preparation method has small coding space and low strength of marked fluorescent dye, and cannot meet the requirement of biochemical detection.
In addition, fluorescent dyes are essentially organic compounds containing unsaturated bonds, which are easily oxidized by oxygen in air or water, resulting in fluorescence quenching. When fluorescent microspheres or fluorescent coding microspheres prepared by the traditional method are exposed to biological samples, aqueous solutions and other samples, because a large amount of oxygen molecules exist in the samples, fluorescence is easily quenched, and the detection of fluorescent signals by an instrument is affected.
Disclosure of Invention
Based on the above, the invention provides a fluorescent microsphere with high fluorescence intensity, a preparation method and a fluorescence coding method thereof.
A preparation method of fluorescent microspheres comprises the following steps:
step one: mixing the microspheres with a silane reagent and a solvent for activation to obtain activated microspheres, wherein the silane reagent is at least one of an epoxy silane reagent, a chlorosilane reagent, an isocyanic acid silane reagent, a succinic anhydride silane reagent and a chlorosulfonyl phenyl silane reagent;
step two: and (3) carrying out coupling reaction on the activated microsphere and the fluorescent dye in a solvent to obtain the fluorescent microsphere.
In one embodiment, the fluorescent dye in the coupling reaction is two or more kinds, and the fluorescent microsphere with multicolor fluorescence is prepared.
In one embodiment, the fluorescent microsphere is further coated with silica.
Aiming at the problem of fluorescence quenching in the traditional preparation method of the fluorescent microsphere, the invention also provides a silicon dioxide coating method of the fluorescent microsphere, which creatively coats silicon dioxide on the surface of the fluorescent microsphere or the fluorescent coding microsphere as a protective layer, thereby avoiding the fluorescent quenching caused by exposing fluorescent dye in biological, aqueous solution and other samples, further improving the stability of the fluorescent intensity of the dye and ensuring the fluorescent intensity of the dye to meet the detection requirement for a long time. In addition, the method is favorable for modification and activation on the surface of the silicon dioxide, and is further used for marking different biological samples.
In one embodiment, the step of coating the fluorescent microsphere with silica is as follows:
and (3) mixing and stirring the fluorescent microspheres, ethanol and polyvinylpyrrolidone, centrifuging and standing to remove supernatant, mixing the solid with ethanol, adding ethyl orthosilicate, and stirring to obtain the silica-coated fluorescent microspheres.
In one embodiment, the method further comprises the steps of replacing the microspheres with silica coated fluorescent microspheres and activating according to the first step, preparing activated fluorescent microspheres, replacing the activated microspheres with the prepared activated fluorescent microspheres, and performing a coupling reaction according to the second step.
In one embodiment, the silane reagent has a methoxysilane structure or an ethoxysilane structure.
In one embodiment, the epoxysilane reagent is 3-glycidoxypropyl triethoxysilane, the chlorosilane reagent is 3-chloropropyl triethoxysilane, the isocyanatosilane reagent is propyltriethoxysilane isocyanate, the succinic anhydride silane reagent is dihydro-3- [3- (triethoxysilyl) propyl ] furan-2, 5-dione, and the chlorosulfonylphenylsilane reagent is 2- (4-chlorosulfonylphenyl) ethyltrimethoxysilane.
In one embodiment, the fluorescent dye is a fluorescent dye that itself has a reactive group capable of coupling with at least one silane reagent or a fluorescent dye modified with a reactive group capable of coupling with at least one silane reagent.
In one embodiment, the mass-to-volume concentration of each fluorescent dye added is controlled to be changed in a gradient manner, so that one or more coded fluorescent microspheres with different fluorescence intensities are prepared.
A preparation method of fluorescent microspheres comprises the following steps:
step one: coating fluorescent microspheres with silicon dioxide, and then mixing the fluorescent microspheres with a silane reagent and a solvent for activation to obtain fluorescent activated microspheres, wherein the silane reagent is at least one of an epoxy silane reagent, a chlorosilane reagent, an isocyanic acid silane reagent, a succinic anhydride silane reagent and a chlorosulfonyl phenyl silane reagent;
step two: and (3) carrying out coupling reaction on the activated microsphere and the fluorescent dye in a solvent to obtain the fluorescent microsphere.
The preparation method of the fluorescent microsphere adopts the silane reagent to directly act with the microsphere under neutral conditions without catalysis of acidic or alkaline conditions, and forms epoxy groups, chlorine groups, isocyanic acid groups, succinic anhydride groups or chlorosulfonyl phenyl groups on the surface of the microsphere, and the problem of poor activation effect caused by ring opening or hydrolysis of the groups under acidic or alkaline conditions is avoided; the prepared active microsphere can directly perform coupling reaction with fluorescent dye without the action of a coupling agent, so as to obtain the fluorescent microsphere. According to the invention, compared with the traditional swelling method or the preparation method of coating the silanized fluorescent dye on the microsphere surface, the fluorescent microsphere prepared by the preparation method has the advantages of large fluorescent dye coding space, high marked fluorescent dye strength and the like, is very convenient for fluorescent coding, avoids the defect that the fluorescent intensity of the fluorescent coding microsphere prepared by the traditional preparation method cannot meet the requirement, and has high fluorescent intensity, thus being capable of meeting the detection requirement.
In one embodiment, the fluorescent dye in the coupling reaction is two or more kinds, and the fluorescent microsphere with multicolor fluorescence is prepared.
In one embodiment, the mass-to-volume concentration of each fluorescent dye added is controlled to be changed in a gradient manner, so that one or more coded fluorescent microspheres with different fluorescence intensities are prepared.
The fluorescent microsphere according to any one of the above methods.
Drawings
FIG. 1 is a photograph of silica coated fluorescent microspheres prepared in example 1 under a microscope;
FIG. 2 shows the results of detection of the APC-encoded fluorescent microspheres of different fluorescence intensities prepared in example 2 in a flow cytometer;
FIG. 3 shows the detection results of fluorescent microspheres of different fluorescence intensities encoded by two fluorescent dyes, PE and APC, prepared in example 6 in a flow cytometer.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention, and preferred embodiments of the present invention are set forth. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The preparation method of the fluorescent microsphere in one embodiment comprises the following steps S1-S2.
Step S1: mixing the microsphere with a silane reagent and a solvent for activation to obtain an activated microsphere; wherein the silane reagent is at least one of epoxy silane reagent, chloro silane reagent, isocyanic acid silane reagent, succinic anhydride silane reagent and chlorosulfonyl phenyl silane reagent.
The silane reagent is adopted, no acid or alkaline condition is needed for catalysis, the silane reagent can directly act with the microsphere to activate the microsphere under neutral condition, and epoxy group, chlorine group, isocyanic acid group, succinic anhydride group or chlorosulfonyl phenyl group is formed on the surface of the microsphere, and the problem of poor activation effect caused by ring opening or hydrolysis of the groups under acid or alkaline condition is avoided; the prepared active microsphere does not need the action of a coupling agent, and can directly perform a coupling reaction with fluorescent dye to obtain the fluorescent microsphere.
The silane reagent has wide selection range of the activation groups, enriches the selection of the activation groups, compensates the defect of less active coupling group types of fluorescent dyes sold in the market at present, and increases the universality of microsphere surface marking.
Further, in step S1, the solvent is water.
Specifically, the activation condition is that mixing is carried out for 0.5-48 h at 18-80 ℃.
In one embodiment, the silane reagent has a methoxysilane structure or an ethoxysilane structure. The silane reagent with methoxysilane structure or ethoxysilane structure is easy to remove methoxy or ethoxy to hydrolyze, so that the coupling reaction can be promoted.
Further, the epoxysilane reagent is 3-glycidoxypropyl triethoxysilane.
Further, the chlorosilane reagent is 3-chloropropyl triethoxysilane.
Further, the isocyanatosilane reagent is propyltriethoxysilane isocyanate.
Further, the succinic anhydride silane reagent is dihydro-3- [3- (triethoxysilyl) propyl ] furan-2, 5-dione.
Further, the chlorosulfonylphenylsilane reagent is 2- (4-chlorosulfonylphenyl) ethyltrimethoxysilane.
Further, it also includes a silane reagent having the same active end as the above and a secondary or multiple silane reagent obtained by bridging coupling or modifying other active groups on the basis of other silane reagents having an oxysilane structure.
Specifically, the mass volume ratio of the microspheres to the silane reagent is (0.01 mg-500 mg): 1mL. Preferably, the mass volume ratio of the microsphere to the silane reagent is (20 mg-30 mg): 1mL.
Further, the microsphere is a high molecular polymer microsphere, a magnetic microsphere or a silicon dioxide microsphere. Specifically, the high molecular polymer microsphere is a polystyrene microsphere, a polymethyl methacrylate microsphere or a polyethylene-methyl methacrylate copolymer microsphere. Specifically, the magnetic microspheres are ferroferric oxide microspheres or ferric oxide microspheres. Specifically, the surface of the magnetic microsphere may be coated with silica or a polymer.
Further, the microspheres in the step S1 are added in the form of microsphere solution, and the mass volume ratio of the microsphere solution is (0.01 mg-500 mg): 1mL. Preferably, the mass volume ratio of the microsphere solution is (20 mg-30 mg): 1mL.
Specifically, the particle size of the microspheres is 2-40 microns. Preferably, the particle size of the microspheres is from 5 to 10 microns.
Further, the stirring speed of the mixing in the step S1 is 50-500 rpm.
Step S2: and (3) carrying out coupling reaction on the activated microsphere and the fluorescent dye in a solvent to obtain the fluorescent microsphere.
Specifically, the condition of the coupling reaction is that the reaction is carried out for 0.5 to 48 hours at the temperature of 18 to 50 ℃. Preferably, the conditions of the coupling reaction are such that the reaction is carried out at 30 to 40℃for 18 to 24 hours.
More specifically, the stirring speed of the step S2 coupling reaction is 50 to 500rpm. Further, in step S2, the solvent is water.
The fluorescent dye may be a fluorescent dye that itself has a reactive group that is capable of coupling with at least one silane reagent; fluorescent dyes modified with reactive groups that can be coupled to at least one silane reagent are also possible. The fluorescent dye also comprises any secondary or multiple fluorescent dye obtained by bridging or modifying other active groups capable of being coupled with a silane reagent.
Further, the fluorescent dye may be at least one of FITC (isocyanic acid fluorescent dye), RBITC (rhodamine), PE (phycoerythrin), APC (allophycocyanin) or tandem fluorescent dye. Wherein FITC and RBITC have isocyanate groups, and PE and APC have amino groups. The fluorescent dye is phycoerythrin, allophycocyanin or serial dye containing at least one of phycoerythrin and allophycocyanin, and can react with any one of the silane reagents. When the fluorescent dye is FITC or RBITC, amino groups need to be modified first, or polyethyleneimine is added during coupling reaction to bridge silane reagent and fluorescent dye.
Further, a fluorescent dye having an amino group is selected. Further, the ratio of the mass of the microsphere corresponding to the active microsphere to the mass of each fluorescent dye is 100 (0.15-10).
In the step S2, two or more fluorescent dyes are adopted to perform coupling reaction with the active microspheres, so that a plurality of signals with different fluorescence can be obtained.
As the activated groups on the activated microsphere are more, the fluorescent dye has large coding space, and the fluorescent intensity can be controlled by controlling the amount of the fluorescent dye, so that the fluorescent intensity can be adjusted, and the microsphere with different fluorescent intensities, namely the single fluorescent coding microsphere, is obtained. If the mass-volume ratio of the fluorescent dyes is controlled to be changed in a gradient way, the coded fluorescent microsphere with multiple fluorescent lights and different intensities is prepared, and the coded fluorescent microsphere with multiple fluorescent lights is obtained. This is the fluorescent coding method of fluorescent microspheres.
That is, the mass-to-volume ratio of each fluorescent dye added is controlled to be changed in a gradient manner, so that one or more coded fluorescent microspheres with different fluorescence intensities are prepared. If the number of the fluorescent dyes added is multiple, different fluorescent intensity signals of multiple different fluorescent lights can be obtained, and the fluorescent microspheres with multiple fluorescent codes can be obtained by integrating the types and the mass-volume ratio changes of the fluorescent dyes.
Preferably, when two or more fluorescent dyes are used, the combination thereof may be a combination of PE and APC, FITC and RBITC, APC and tandem fluorescent dye APC-Cy 7. The preparation method has the advantage of large coding space of the fluorescent dye, so that multiple fluorescent dyes can be simultaneously coded, and the prepared fluorescent coding microsphere has high fluorescent intensity. Specifically, the mass ratio of PE to APC is 100: (0.01-10000). More specifically, the mass ratio of PE to APC is 100 (20 to 200).
Specifically, 1mg of PE was dissolved in 1mL of deionized water to obtain a PE dye solution. APC was dissolved in 1mL deionized water to obtain an APC dye solution. PE and APC are combined according to (80,120), (80, 100), (100 ) (80, 80), (80, 110), (60, 80), (40, 80), (30, 80), (60, 60), (120, 60), (110,50), (80,50), (60, 40), (70,40), (60, 30) and (50, 50); wherein PE and APC as in (80,120) refer to the addition of 80. Mu.L of PE solution, 120. Mu.L of APC solution, and the like.
According to the invention, compared with the traditional swelling method or the preparation method of coating the silanized fluorescent dye on the microsphere surface, the fluorescent microsphere prepared by the preparation method has the advantages of large fluorescent dye coding space, high marked fluorescent dye strength and the like, and the defect that the fluorescent intensity of the fluorescent coding microsphere prepared by the traditional preparation method cannot meet the requirement is overcome, and the prepared fluorescent coding microsphere has high fluorescent intensity.
In addition, compared with a swelling method, the preparation method has the advantages that the microsphere carrier is not easy to adhere, the size is uniform, and the distribution is uniform.
In one embodiment, the method further comprises the step of coating the fluorescent microspheres or fluorescent-encoded microspheres with silica. Fluorescent dyes are essentially organic compounds containing unsaturated bonds, which are easily oxidized by oxygen in air or water, resulting in fluorescence quenching. Therefore, the invention creatively coats silicon dioxide on the surface of the fluorescent microsphere or the fluorescent coding microsphere as a protective layer, and avoids exposing fluorescent dye in biological, aqueous solution and other samples, thereby avoiding quenching of fluorescence caused by a large amount of oxygen in the samples, further affecting the detection of fluorescent signals by an instrument, further improving the stability of dye fluorescence, and ensuring that the dye fluorescence meets the detection requirement for a long time. In addition, the method is also beneficial to modifying an activated silane reagent on the surface of the silicon dioxide for marking different biological samples.
Specifically, the step of coating the fluorescent microsphere or the fluorescent coding microsphere with silicon dioxide comprises the following steps: stirring fluorescent microspheres or fluorescent coding microspheres, ethanol and polyvinylpyrrolidone (PVP) for 0.5-48 h at 20-70 ℃, standing to remove supernatant, mixing the solid with ethanol, adding Tetraethoxysilane (TEOS) and stirring for 0.5-48 h at 20-70 ℃ to obtain the silica coated fluorescent microspheres or silica coated fluorescent coding microspheres. PVP is used as a surfactant, has good affinity with silicon dioxide and fluorescent dye, and is favorable for coating the silicon dioxide.
Preferably, stirring the fluorescent microsphere or fluorescent coding microsphere, ethanol and polyvinylpyrrolidone for 2-4 hours at 30-50 ℃, standing to remove supernatant, mixing the solid with ethanol, adding tetraethoxysilane and ammonia water, and stirring for 2-4 hours at 30-50 ℃ to obtain the silica coated fluorescent microsphere or fluorescent coding microsphere.
Specifically, the ratio of the mass of the fluorescent microsphere or the fluorescent coding microsphere corresponding to the raw material microsphere to the mass of polyvinylpyrrolidone is 0.5-4:1. Preferably, the ratio of the mass of the fluorescent microsphere or the fluorescent coding microsphere corresponding to the raw material microsphere to the mass of polyvinylpyrrolidone is 2:1.
Specifically, the ratio of the mass of the raw material microspheres corresponding to the fluorescent microspheres or the fluorescent coded microspheres to the volume of the tetraethoxysilane is (10 mg-30 mg): 1mL. Preferably, the ratio of the mass of the fluorescent microsphere or the fluorescent coding microsphere corresponding to the raw material microsphere to the volume of the tetraethoxysilane is 20 mg/1 mL.
Specifically, the ratio of the mass of the fluorescent microsphere or the fluorescent coded microsphere corresponding to the raw material microsphere to the volume of the ammonia water is (10 mg-30 mg): 1mL. Preferably, the ratio of the mass of the fluorescent coding microsphere corresponding to the raw material microsphere to the volume of ammonia water is 20 mg/1 mL.
Specifically, when the microspheres are magnetic microspheres, the supernatant may be removed by magnetic separation to separate the solids from the solvent.
Further, the method also comprises the steps of replacing the microsphere in the step S1 by a microsphere coated with silicon dioxide, activating according to the step S1 to obtain an activated microsphere coated with silicon dioxide, and performing a coupling reaction according to the step S2. Specifically, the steps of the activation and coupling reaction may be the same as those of steps S1 to S2, or may not be limited to the above-mentioned raw materials.
The invention also provides a preparation method of the fluorescent microsphere in an embodiment. The preparation method adopts the microsphere with fluorescence, and the step S1 and the step S2 are carried out after the silica is coated firstly to replace the microsphere in the preparation method.
It is understood that the microsphere with fluorescence can be the microsphere with fluorescence prepared by any step of the preparation method of the fluorescent microsphere, and can also be the microsphere with fluorescence prepared by the existing or other methods.
It will be appreciated that the steps S1, S2 and other steps in this embodiment may be similar to the preparation methods described above. For example: the fluorescent dye in the coupling reaction is two or more kinds, and the fluorescent microsphere with multicolor fluorescence is prepared. For example: the mass volume ratio concentration of each added fluorescent dye is controlled to be changed in a gradient way, so that one or more coded fluorescent microspheres with different fluorescence intensities are prepared.
Further, the invention also provides the fluorescent coding microsphere prepared by the preparation method of the fluorescent coding microsphere in any embodiment.
The fluorescent coding microsphere prepared by the preparation method has the advantages of large fluorescent dye coding space, high marked fluorescent dye strength and the like.
The following are specific examples.
Example 1
Activation of microsphere surface: into a 250mL three-necked flask, 30mL deionized water and 1mL 20mg/mL Fe were added 3 O 4 And (3) stirring the microsphere solution and 1mL of epoxy silane reagent (3-glycidoxypropyl triethoxysilane) for 24 hours at the rotating speed of 200rpm and the temperature of 50 ℃ to obtain the epoxy modified activated microsphere. Fe (Fe) 3 O 4 The particle size of the microspheres was 5 microns.
Microsphere coupled fluorescent dye: 30mL of deionized water, the epoxy modified activated microsphere and 80 mu L of PE fluorescent dye with the concentration of 1mg/mL are added into a 250mL three-neck flask, and the mixture is stirred for 24 hours at the speed of 200rpm and the temperature of 30 ℃ to obtain the microsphere with PE fluorescence.
Example 2
Substantially the same as in example 1, except that the fluorescent dye was APC, that is, 10. Mu.L, 30. Mu.L, 60. Mu.L, 120. Mu.L of an APC dye solution of 1mg/mL was added, microspheres having four different APC fluorescence intensities were obtained.
Examples 3 and 4
Examples 3 and 4 are substantially the same as examples 1 and 2 except that Fe 3 O 4 The concentration of the microsphere solution is 10mg/mL and 30mg/mL respectively, and the microsphere solution is also in different Fe 3 O 4 Under the condition of microsphere solution concentration, the microsphere with PE or APC fluorescence can be obtained.
Example 5
Substantially the same as in example 1, except that the PE fluorescent microspheres obtained in example 1 were silica-coated, the procedure was as follows:
adding the fluorescent microsphere into a 250mL three-neck flask, adding 30mL of ethanol and 10mg of polyvinylpyrrolidone (PVP), stirring for 2 hours at 30 ℃, magnetically separating, redissolving into 30mL of ethanol, adding 1mL of tetraethyl orthosilicate TEOS, and stirring for 2 hours at 30 ℃ at a rotating speed of 200rpm to obtain the corresponding silica coated fluorescent microsphere.
Example 6
Activation of microsphere surface: into a 250mL three-necked flask, 30mL deionized water and 1mL 20mg/mL Fe were added 3 O 4 And (3) stirring the microsphere solution and 1mL of epoxy silane reagent (3-glycidoxypropyl triethoxysilane) for 24 hours at the rotating speed of 200rpm and the temperature of 50 ℃ to obtain the epoxy modified activated microsphere. Fe (Fe) 3 O 4 The particle size of the microspheres was 5 microns.
Microsphere coupled fluorescent dye: 30mL of deionized water, the epoxy modified activated microsphere, PE and APC are added into a 250mL three-neck flask, and stirred for 24 hours at the rotating speed of 200rpm and the temperature of 30 ℃ to obtain the fluorescent dye PE and APC microsphere. And combining PE and APC fluorescent dye according to different masses, and respectively performing a step of coupling the fluorescent dye to obtain the encoded microsphere with different PE and APC fluorescent intensity. The proportions of PE and APC fluorescent dye combined by different mass-volume ratios are as follows:
1mg of PE fluorescent dye is dissolved in 1mL of deionized water to obtain PE dye solution. APC was dissolved in 1mL deionized water to obtain an APC dye solution. PE and APC are combined according to (80,120), (80, 100), (100 ), (80, 80), (80, 110), (60, 80), (40, 80), (30, 80), (60, 60), (120, 60), (110,50), (80,50), (60, 40), (70,40), (60, 30) and (50, 50), wherein PE and APC refer to the addition of 80. Mu.L of PE fluorochrome solution, 120. Mu.L of APC fluorochrome solution, and the like according to (80,120).
Example 7
Activation of microsphere surface: into a 250mL three-necked flask, 30mL deionized water and 1mL 20mg/mL Fe were added 3 O 4 And stirring the microsphere solution and 1mL of chlorosilane reagent (3-chloropropyl triethoxysilane) for 24 hours at the rotating speed of 200rpm and the temperature of 50 ℃ to obtain the chloro-modified activated microsphere.
Microsphere coupled fluorescent dye: 30mL of deionized water, the chlorinated modified activated microsphere, PE and APC fluorescent dye are added into a 250mL three-neck flask, and stirred for 24 hours at the speed of 200rpm and the temperature of 30 ℃ to obtain PE and APC fluorescent dye microspheres. And respectively carrying out the step of coupling the PE and the APC fluorescent dye according to different combinations to obtain the encoded microsphere with different PE and APC fluorescent intensity. The proportions of PE and APC fluorochromes combined in different mass to volume ratios were the same as in example 5.
Example 8
Activation of microsphere surface: into a 250mL three-necked flask, 30mL deionized water and 1mL 20mg/mL Fe were added 3 O 4 And stirring the microsphere solution and 1mL of isocyanatosilane reagent (isocyanatopropyl triethoxysilane) for 24 hours at the rotating speed of 200rpm and the temperature of 50 ℃ to obtain the isocyanic acid modified activated microsphere.
Microsphere coupled fluorescent dye: adding 30mL of deionized water, the isocyanate modified activated microsphere, PE and APC fluorescent dye into a 250mL three-neck flask, and stirring for 24h at the speed of 200rpm and the temperature of 30 ℃ to obtain PE and APC fluorescent microsphere. And combining PE and APC fluorescent dye according to different masses, and respectively performing a step of coupling the fluorescent dye to obtain the encoded microsphere with different PE and APC fluorescent intensity. The proportions of the PE and APC fluorescent dyes were the same as in example 6.
Example 9
Activation of microsphere surface: into a 250mL three-necked flask, 30mL deionized water and 1mL 20mg/mL Fe were added 3 O 4 Microsphere solution and 1mL succinic anhydride silane reagent (dihydro-3- [3- (triethoxysilyl) propyl)]Furan-2, 5-dione) and stirring for 20 hours at the speed of 200rpm and the temperature of 60 ℃ to obtain succinic anhydride modified activated microspheres.
Microsphere coupled fluorescent dye: adding 30mL of deionized water, the succinic anhydride modified activated microsphere, PE and APC fluorescent dye into a 250mL three-neck flask, and stirring for 18h at the speed of 200rpm and the temperature of 40 ℃ to obtain the PE and APC fluorescent microsphere. And combining PE and APC according to different masses, and respectively performing a step of coupling fluorescent dyes to obtain the encoded microspheres with different PE and APC fluorescent intensities. The proportions of the PE and APC fluorescent dyes were the same as in example 6.
Example 10
Activation of microsphere surface: into a 250mL three-necked flask, 30mL deionized water and 1mL 20mg/mL Fe were added 3 O 4 And (3) stirring the microsphere solution and 1mL of chlorosulfonylphenyl silane reagent (2- (4-chlorosulfonylphenyl) ethyltrimethoxysilane) at the rotating speed of 200rpm and the temperature of 70 ℃ for 18 hours to obtain the chlorosulfonylphenyl modified activated microsphere.
Microsphere coupled fluorescent dye: 30mL of deionized water, the chlorosulfonyl phenyl modified activated microsphere, PE and APC fluorescent dye are added into a 250mL three-neck flask, and stirred for 20h at the speed of 200rpm and the temperature of 35 ℃ to obtain the PE and APC fluorescent microsphere. And combining PE and APC fluorescent dye according to different masses, and respectively performing a step of coupling the fluorescent dye to obtain the encoded microsphere with different PE and APC fluorescent intensity. The proportions of the PE and APC fluorescent dyes were the same as in example 6.
Example 11
Activation of microsphere surface: into a 250mL three-necked flask, 30mL deionized water and 1mL 20mg/mL Fe were added 3 O 4 And (3) stirring the microsphere solution and 1mL of epoxy silane reagent (3-glycidoxypropyl triethoxysilane) for 24 hours at the rotating speed of 200rpm and the temperature of 50 ℃ to obtain the epoxy modified activated microsphere.
Microsphere coupled fluorescent dye: 30mL of deionized water, the epoxy modified activated microsphere, FITC and RBITC fluorescent dye and 50mg of Polyethylenimine (PEI) are added into a 250mL three-neck flask, and stirred for 24 hours at the speed of 200rpm and the temperature of 30 ℃ to obtain the FITC and RBITC fluorescent microsphere. And combining FITC and RBITC fluorescent dyes according to different masses, and respectively performing a step of coupling the fluorescent dyes to obtain the encoded microspheres with different FITC and RBITC fluorescent intensities. The proportions of the different mass combinations FITC and RBITC fluorescent dyes were the same as in example 6.
Example 12
Activation of microsphere surface: into a 250mL three-necked flask, 30mL deionized water and 1mL 20mg/mL Fe were added 3 O 4 Microsphere solution and 1mL of epoxysilane reagent (3-glycidoxypropyl triethoxysilane) at 200rpm and 50 DEG CStirring for 24h to obtain the epoxy modified activated microsphere.
Microsphere coupled fluorescent dye: 30mL of deionized water, the epoxy modified activated microsphere, the APC and the APC-Cy7 tandem fluorescent dye are added into a 250mL three-neck flask, and stirred for 24 hours at the speed of 200rpm and the temperature of 30 ℃ to obtain the APC and the APC-Cy7 fluorescent microsphere. And (3) combining the APC and the APC-Cy7 fluorescent dye according to different masses, and respectively performing a step of coupling the fluorescent dye to obtain the encoded microsphere with different APC and APC-Cy7 fluorescent intensities. The ratios of the APC and APC-Cy7 fluorescent dyes of different mass combinations were the same as in example 6.
Example 13
In examples 1 to 4 and examples 6 to 12, silica coating was performed in the same manner as in example 5 to obtain corresponding silica-coated fluorescent encoded microspheres.
The performance test is as follows:
the silica-coated fluorescent microsphere prepared in example 1 was observed under a microscope, and a photograph was obtained as shown in FIG. 1. As can be seen from FIG. 1, the silica-coated fluorescent encoded microspheres are non-sticky, uniform in size and uniform in distribution. The photographs under a microscope of the silica-uncoated fluorescent microspheres or fluorescent-encoded microspheres prepared in examples 2 to 12 and the silica-coated fluorescent-encoded microspheres prepared in examples 5 and 13 were similar to those of fig. 1.
The detection results of the fluorescent microspheres of different fluorescence intensities encoded by APC prepared in example 2 in a flow cytometer are shown in fig. 2. The abscissa FSC refers to the forward scatter (Forward Scattering) of the microspheres in the flow cytometer, which is directly related to the relative size of the particle size of the microspheres, and the ordinate is the corresponding APC fluorescence intensity of the fluorescent microspheres as measured by the flow cytometer. It can be seen that the fluorescent microsphere prepared by the method has very aggregated clusters and uniform fluorescence intensity compared with the known swelling method, and can be used for coding a larger fluorescence space.
The detection results of the fluorescent microspheres of different fluorescence intensities encoded by two fluorescent dyes, PE and APC, prepared in example 6 in a flow cytometer are shown in FIG. 3. The abscissa and the ordinate are the fluorescence intensities of PE and APC, respectively, measured by flow cytometry of the fluorescent microspheres. It can also be seen that the two-dimensional fluorescent coded micro-pellet clusters are still very aggregated, have uniform fluorescent intensity and have very large fluorescent coding space.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (11)

1. The preparation method of the fluorescent microsphere is characterized by comprising the following steps:
step one: mixing the microspheres with a silane reagent and a solvent for activation to obtain activated microspheres, wherein the silane reagent is at least one of an epoxy silane reagent, a chlorosilane reagent, an isocyanic acid silane reagent, a succinic anhydride silane reagent and a chlorosulfonyl phenyl silane reagent;
step two: coupling the activated microsphere and the fluorescent dye in a solvent to obtain the fluorescent microsphere; the fluorescent dye is at least one of FITC, RBITC, PE, APC and serial fluorescent dye;
the preparation method further comprises the steps of coating the fluorescent microspheres with silicon dioxide, replacing the microspheres in the first step with the fluorescent microspheres coated with the silicon dioxide and activating according to the first step to prepare activated fluorescent microspheres, replacing the activated microspheres in the second step with the activated fluorescent microspheres obtained by preparation, and carrying out a coupling reaction according to the second step.
2. The method of claim 1, wherein the fluorescent dye is two or more kinds of fluorescent dye in the coupling reaction, and the fluorescent microsphere with multicolor fluorescence is obtained.
3. The method of preparing fluorescent microspheres according to claim 1, wherein the step of coating the fluorescent microspheres with silica comprises the steps of:
and (3) mixing and stirring the fluorescent microspheres, ethanol and polyvinylpyrrolidone, centrifuging and standing to remove supernatant, mixing the solid with ethanol, adding ethyl orthosilicate, and stirring to obtain the silica-coated fluorescent microspheres.
4. The method of preparing fluorescent microspheres according to claim 1 or 2, wherein the silane reagent has a methoxysilane structure or an ethoxysilane structure.
5. The method for preparing fluorescent microspheres according to claim 1 or 2, wherein the epoxysilane reagent is 3-glycidoxypropyl triethoxysilane, the chlorosilane reagent is 3-chloropropyl triethoxysilane, the isocyanatosilane reagent is isocyanatopropyl triethoxysilane, the succinic anhydride silane reagent is dihydro-3- [3- (triethoxysilyl) propyl ] furan-2, 5-dione, and the chlorosulfonylphenylsilane reagent is 2- (4-chlorosulfonylphenyl) ethyltrimethoxysilane.
6. The method of preparing a fluorescent microsphere according to claim 1 or 2, wherein the fluorescent dye is a fluorescent dye itself having a reactive group capable of coupling with at least one silane agent or a fluorescent dye modified with a reactive group capable of coupling with at least one silane agent.
7. The method of claim 1 or 2, wherein the mass-to-volume concentration of each fluorescent dye added is controlled to be changed in a gradient manner, so that one or more coded fluorescent microspheres with different fluorescence intensities are prepared.
8. The preparation method of the fluorescent microsphere is characterized by comprising the following steps:
step one: coating fluorescent microspheres with silicon dioxide, and then mixing the fluorescent microspheres with a silane reagent and a solvent for activation to obtain fluorescent activated microspheres, wherein the silane reagent is at least one of an epoxy silane reagent, a chlorosilane reagent, an isocyanic acid silane reagent, a succinic anhydride silane reagent and a chlorosulfonyl phenyl silane reagent;
step two: and (3) carrying out coupling reaction on the activated microsphere and the fluorescent dye in a solvent to obtain the fluorescent microsphere.
9. The method of claim 8, wherein the fluorescent dye is two or more kinds of fluorescent dye in the coupling reaction, and the fluorescent microsphere with multicolor fluorescence is obtained.
10. The method of claim 8 or 9, wherein the mass-to-volume concentration of each fluorescent dye added is controlled to be changed in a gradient manner, so that one or more coded fluorescent microspheres with different fluorescence intensities are prepared.
11. A method of producing a fluorescent microsphere according to any one of claims 1 to 7 or a fluorescent microsphere produced by a method of producing a fluorescent microsphere according to any one of claims 8 to 10.
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