CN115094644A - Preparation method and application of bicolor microspheres - Google Patents
Preparation method and application of bicolor microspheres Download PDFInfo
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- CN115094644A CN115094644A CN202210765919.4A CN202210765919A CN115094644A CN 115094644 A CN115094644 A CN 115094644A CN 202210765919 A CN202210765919 A CN 202210765919A CN 115094644 A CN115094644 A CN 115094644A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 44
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- 239000011259 mixed solution Substances 0.000 claims abstract description 76
- 229920002223 polystyrene Polymers 0.000 claims abstract description 70
- DGEZNRSVGBDHLK-UHFFFAOYSA-N [1,10]phenanthroline Chemical compound C1=CN=C2C3=NC=CC=C3C=CC2=C1 DGEZNRSVGBDHLK-UHFFFAOYSA-N 0.000 claims abstract description 54
- YNPNZTXNASCQKK-UHFFFAOYSA-N Phenanthrene Natural products C1=CC=C2C3=CC=CC=C3C=CC2=C1 YNPNZTXNASCQKK-UHFFFAOYSA-N 0.000 claims abstract description 53
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- GMQVFHZSXKJCIV-UHFFFAOYSA-N 2,2,2-trifluoro-n-(2,2,2-trifluoroacetyl)acetamide Chemical class FC(F)(F)C(=O)NC(=O)C(F)(F)F GMQVFHZSXKJCIV-UHFFFAOYSA-N 0.000 description 3
- 229910052693 Europium Inorganic materials 0.000 description 3
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 3
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- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 2
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- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 1
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- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06P—DYEING OR PRINTING TEXTILES; DYEING LEATHER, FURS OR SOLID MACROMOLECULAR SUBSTANCES IN ANY FORM
- D06P1/00—General processes of dyeing or printing textiles, or general processes of dyeing leather, furs, or solid macromolecular substances in any form, classified according to the dyes, pigments, or auxiliary substances employed
- D06P1/0004—General aspects of dyeing
- D06P1/0012—Effecting dyeing to obtain luminescent or phosphorescent dyeings
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- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06P—DYEING OR PRINTING TEXTILES; DYEING LEATHER, FURS OR SOLID MACROMOLECULAR SUBSTANCES IN ANY FORM
- D06P1/00—General processes of dyeing or printing textiles, or general processes of dyeing leather, furs, or solid macromolecular substances in any form, classified according to the dyes, pigments, or auxiliary substances employed
- D06P1/0004—General aspects of dyeing
- D06P1/002—Processing by repeated dyeing, e.g. in different baths
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- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06P—DYEING OR PRINTING TEXTILES; DYEING LEATHER, FURS OR SOLID MACROMOLECULAR SUBSTANCES IN ANY FORM
- D06P3/00—Special processes of dyeing or printing textiles, or dyeing leather, furs, or solid macromolecular substances in any form, classified according to the material treated
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
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Abstract
The invention relates to a preparation method of bicolor microspheres, which comprises the following steps: preparation of S1 fluorescent dye: respectively preparing a DBM and ethanol mixed solution, a Phen and ethanol mixed solution and a rare earth ion and ethanol mixed solution according to a ratio, reacting the DBM and ethanol mixed solution and the Phen and ethanol mixed solution with the rare earth ion and ethanol mixed solution, and filtering and drying to obtain fluorescent dye powder; preparation of S2 polystyrene microsphere: preparing polystyrene microspheres by a soap-free emulsion polymerization method to obtain polystyrene microsphere liquid; s3 preparation of polystyrene color microspheres: mixing the color dye and polystyrene microsphere liquid, emulsifying, centrifuging, and removing supernatant to obtain polystyrene color microspheres; s4 preparation of polystyrene PS bicolor microspheres: and (4) mixing the fluorescent dye powder prepared in the step (S1) with the polystyrene colorful microspheres prepared in the step (S3), emulsifying, and centrifuging to remove supernatant liquid to obtain the polystyrene PS double-color microspheres.
Description
Technical Field
The invention relates to the technical field of fluorescence immunoassay, and relates to a preparation method and application of a bicolor microsphere.
Background
The polymer microsphere has the characteristics of stable morphological structure, narrow particle size distribution, large specific surface area, strong surface adsorbability and the like, and is widely applied to the fields of life science, biomedicine and the like, such as cell surface antigen detection, blood flow analysis, phagocytic function detection, diagnostic detection represented by agglutination test, drug screening and the like.
The polymer microsphere is white, has low contrast and is difficult to detect, but when the microsphere is subjected to dye and fluorescence labeling and becomes the microsphere with color and fluorescence signals, the detection is easy. Particularly, for a latex agglutination test based on polystyrene microspheres, the colored microspheres are easier to observe, so that the detection sensitivity is greatly improved.
Chinese patent document (application number: 202110934220.1) discloses a monodisperse polymer color microsphere and a preparation method and application thereof, wherein the preparation method comprises the following steps: (1) preparing carboxyl polystyrene microspheres by adopting a soap-free emulsion polymerization method; (2) providing a swelling medium system with a dissolved dye; (3) and (3) permeating the dye into the interior of the carboxyl microsphere by a seed swelling method to obtain the colorful carboxyl polystyrene microsphere with the dye wrapped in the interior of the microsphere. The method adopts a one-step swelling method to obtain the functional microspheres embedded with the color dye, the seed microspheres used for swelling are carboxyl latex microspheres obtained in a soap-free emulsion polymerization mode, the operation is simple, the size of the obtained microspheres is uniform and controllable, and the surfaces of the microspheres are regular; can be used for immunochromatography detection test strip application. But the dye color microsphere prepared by the method.
Chinese patent document (application number: 202010433079.2) discloses a preparation method of carboxylated polystyrene fluorescent microspheres, which specifically comprises the following steps: preparation of fluorescent substance-europium complex: will dissolve EuCl 3 ·6H 2 Adding ethanol solution of O into ethanol solution of TPPO for coordination reaction, adding deprotonated BTFA ethanol solution into Eu (TPPO) 4 Performing coordination reaction at high temperature, filtering, volatilizing, crystallizing to obtain europium complex Eu (BTFA) (TPPO) 3 (ii) a Utilizing the obtained europium complex Eu (BTFA) (TPPO) 3 Preparing fluorescent polystyrene microsphere and polymerizingAnd performing carboxylation treatment on the surface of the styrene fluorescent microsphere to obtain the polystyrene fluorescent microsphere with the surface containing carboxyl monomers. However, the method prepares single fluorescent microspheres.
Disclosure of Invention
The invention aims to provide a preparation method of bicolor microspheres, which can be used for qualitatively observing by eyes during immunochromatography detection and analysis by simultaneously dyeing the microspheres by fluorescent dye and colored dye and detecting fluorescent signals by an instrument, has more flexible detection and wider application range, and greatly improves the detection sensitivity.
In order to solve the technical problems, the invention adopts the technical scheme that the preparation method of the bicolor microsphere comprises the following steps:
preparation of S1 fluorescent dye: respectively preparing a DBM (dibenzoylmethane) and ethanol mixed solution, a Phen (phenanthroline) and ethanol mixed solution and a rare earth ion and ethanol mixed solution according to a proportion, reacting the DBM and ethanol mixed solution, the Phen and ethanol mixed solution and the rare earth ion and ethanol mixed solution, and performing suction filtration and drying to obtain fluorescent dye powder;
preparation of S2 polystyrene microspheres: preparing polystyrene microspheres by a soap-free emulsion polymerization method to obtain white emulsion, namely polystyrene microsphere liquid (PSt);
s3 preparation of polystyrene color microspheres: mixing the polystyrene microsphere solution obtained in the step S2 with a color dye, emulsifying, and centrifuging to remove supernatant to obtain polystyrene color microspheres;
s4 preparation of polystyrene PS bicolor microspheres: and (3) mixing the fluorescent dye powder prepared in the step S1 with the polystyrene colorful microspheres prepared in the step S3, emulsifying, and centrifuging to remove supernatant to obtain the polystyrene PS double-color microspheres.
As a preferred embodiment of the present invention, the step S1 includes:
s11: weighing raw materials, including Eu 3+ Salt, DBM and Phen for standby;
s12: respectively dissolving DBM and Phen in absolute ethyl alcohol to obtain DBMMixing with ethanol solution and mixed solution of Phen and ethanol, adding Eu 3+ Dissolving salt in absolute ethyl alcohol to obtain an absolute ethyl alcohol mixed solution of rare earth ions;
s13: adding the DBM and ethanol mixed solution into an anhydrous ethanol solution of rare earth ions, placing the mixture into a three-neck flask, heating the mixture at 60-70 ℃, carrying out reflux stirring reaction, dropwise adding the Phen and ethanol mixed solution into the three-neck flask by using a constant-pressure dropping funnel, continuously reacting, adjusting the pH value of the reaction solution, and continuously carrying out reflux stirring reaction until the reaction is finished;
s14: transferring the reaction solution into a clean open container, standing at room temperature, cooling with ice water bath, vacuum filtering, and drying to obtain Eu (DBM) 3 Phen fluorescent dye powder.
As a preferred technical solution of the present invention, the step S2 specifically includes:
s21: adding 200mL of water and 10mL of refined styrene into a three-neck flask, introducing nitrogen, heating in a water bath, and stirring;
s22: and (3) when the temperature of the water bath is raised to 70-80 ℃, adding an initiator Ammonium Persulfate (APS), and continuously stirring for reaction for 20 hours to obtain a uniform white emulsion, namely the polystyrene microsphere solution (PSt). Ammonium persulfate was formulated as 100mg ammonium persulfate dissolved in 5mL of purified water.
As a preferred embodiment of the present invention, the step S3 specifically includes:
s31: dispersing the polystyrene microsphere liquid obtained in the step S2 in a swelling medium; dissolving an oil-soluble yellow R dye in a swelling medium to obtain a dye swelling medium mixed solution;
s32: mixing the microsphere solution obtained in the step S31 with a dye swelling medium mixed solution, and emulsifying, namely carrying out oscillation reaction for 1-3 h at room temperature;
s33: removing the swelling medium by rotary evaporation of a rotary evaporator, performing ultrasonic treatment, gradually diluting the residual liquid, centrifuging, and observing whether the centrifuged precipitate is complete and the fluorescence intensity of the supernatant; discarding the supernatant, washing the lower-layer solid precipitate for multiple times by using pure water, removing the swelling medium solution, washing for multiple times by using the pure water until no color residue exists in the supernatant after centrifugation, removing the oil-soluble yellow R dye which does not enter the interior of the microsphere, and finally dissolving the obtained precipitate in 500 mu L of pure water to obtain the PS colorful microsphere solution. Wherein the swelling medium is 50% acetone aqueous solution.
As a preferred technical solution of the present invention, the step S4 specifically includes:
s41: dispersing the PS colored microsphere liquid (namely 5 mg-10 mg) obtained in the step S3 in 500 mu L of swelling medium; eu (DBM) 3 Dissolving Phen fluorescent dye powder in a swelling medium to obtain a fluorescent dye swelling medium mixed solution;
s42: mixing the microsphere solution and the mixed solution of the fluorescent dye swelling medium in the step S541, emulsifying, namely oscillating reaction at room temperature,
s43: removing the swelling medium by rotary evaporation of a rotary evaporator, performing ultrasonic treatment, gradually diluting the residual liquid, centrifuging, and observing whether the centrifuged precipitate is complete and the fluorescence intensity of the supernatant; discarding the supernatant, washing the lower layer solid precipitate with pure water for several times, removing the swelling medium solution, washing with pure water for several times until the supernatant has no fluorescence after centrifugation, and removing Eu (DBM) not entering into the microsphere 3 Phen, and finally dissolving the obtained precipitate in 500 mu L of pure water to obtain the PS bicolor microsphere solution. Wherein the swelling medium is 50% acetone aqueous solution.
In a preferred embodiment of the present invention, the ratio of the amounts of the substances in step S11 is: eu (Eu) 3+ Weighing raw materials in a ratio of DBM to Phen to 1:3: 1; in the step S13, heating at 60-70 ℃, carrying out reflux stirring reaction for 0.5-1.5 h, dropwise adding a mixed solution of Phen and ethanol into a three-neck flask by using a constant-pressure dropping funnel, continuously reacting for 20-40 min, adjusting the pH value of the reaction solution to 6-7, and continuously heating, carrying out reflux stirring reaction for 3-5 h until the reaction is finished; in the step S14, transferring the reaction solution into a clean open container, standing at room temperature for 24 hours, cooling with ice water bath for 20-40 min, performing suction filtration, and drying the filter cake at 60-70 ℃ to obtain Eu (DBM) 3 Phen fluorescent dye powder.
In a preferred embodiment of the present invention, the three-necked flask in step S21 has a capacity of 500mL, and includes a condenser tube and a mechanical stirrer.
As a preferable technical scheme of the invention, 50-100 mul of polystyrene microsphere solution with the mass fraction of 10% is weighed in step S31 and dispersed in a swelling medium, and 4-5.5 mg of oil-soluble yellow R dye is dissolved in 70-85 mul of the swelling medium; mixing the microsphere solution obtained in the step S31 and the mixed solution of the dye swelling medium in the step S32, and then carrying out ultrasonic treatment for 0-15 min, preferably 5 min; carrying out ultrasonic treatment for 0-10 min every 1-2 h in the emulsification reaction; preferably, every 1h, performing ultrasonic treatment for 3 min; the time of ultrasonic treatment in the step S33 is 1-15 min, preferably 5 min.
As a preferred technical scheme of the invention, 50 to 100 mul of polystyrene colorful microsphere solution with the mass fraction of 10 percent is weighed in the step S41 and is dispersed in a swelling medium; 4 to 5.5mg of Eu (DBM) 3 Dissolving Phen fluorescent dye powder in 70-85 mu L of swelling medium; in the step S42, the microsphere solution and the dye swelling medium mixed solution in the step S41 are mixed and then subjected to ultrasonic treatment, the ultrasonic treatment time is 0-15 min, preferably 5min, and ultrasonic treatment is performed for 0-10 min every 1-2 h in the emulsification reaction; preferably, the ultrasonic treatment is carried out for 3min every 1 h; the time of ultrasonic treatment in the step S43 is 1-15 min, preferably 5 min.
By adopting the technical scheme of the invention, oil-soluble yellow R is respectively taken as a color dye, Eu (DBM) 3 Phen is a fluorescent dye, wherein a fluorescent dye complex Eu (DBM) is adopted 3 The Phen has high fluorescence intensity and low preparation cost; successfully preparing the surface carboxyl functionalized polystyrene microspheres doubly marked by the colored dye and the fluorescent material through twice swelling and embedding; the microspheres are orange yellow under the condition of no excitation light, and emit red fluorescence under the excitation of 360nm ultraviolet rays; the fluorescent dye and the color dye are simultaneously dyed into the microsphere, and the fluorescent dye can be qualitatively seen by eyes on a chromatographic reagent and can also be used for detecting fluorescence by an instrument, so that the detection sensitivity is greatly improved.
As a preferred embodiment of the present invention, a functional group is introduced in the step S2 or the step S3 or the step S4 for coupling an antigen or/and an antibody. The functional group can be hydroxyl and is used for coupling biomolecules such as antigens and antibodies, so that the functional group can be further used for detection and analysis of biological samples and the detection sensitivity is improved.
The bicolor microsphere is applied to a microsphere marker in immunochromatography detection.
Compared with the prior art, the invention has the beneficial effects that: through the fluorescent dye and the color dye for simultaneously dyeing the polystyrene microspheres, the qualitative observation by eyes can be realized during the immunochromatography detection and analysis, and the fluorescent signal can be detected by an instrument, so that the detection is more flexible, the application range is wider, and the detection sensitivity is greatly improved.
Drawings
FIG. 1 is an SEM scanning electron microscope image of a bicolor microsphere obtained by the preparation method of the bicolor microsphere of the invention;
FIG. 2 is a graph showing the results of particle size analysis using a particle size analyzer of the bichromal microspheres obtained by the method for preparing the bichromal microspheres of the invention in example 2;
FIG. 3 is a graph showing a particle size distribution of the bichromal microspheres obtained by the method for preparing the bichromal microspheres of the invention in example 2;
fig. 4 is a structural diagram of a reagent strip assembly of a color comparison experiment of the self-made two-color microspheres obtained by the preparation method of the two-color microspheres of the invention and commercially available single-color colored microspheres in example 3;
FIG. 5 is a comparison chart of the results of the test performed by the reagent strip assembled by the two-color microsphere obtained by the method for preparing the two-color microsphere of the invention and the commercially available single-color colored microsphere in example 3; wherein (a) is the color development result of the reagent strip of the two-color microsphere prepared by the invention, and (b) is the color development result of the reagent strip of the single-color microsphere sold in the market.
Detailed Description
The embodiment is as follows: the preparation method of the bicolor microsphere comprises the following steps:
preparation of S1 fluorescent dye: respectively preparing a DBM and ethanol mixed solution, a Phen and ethanol mixed solution and a rare earth ion and ethanol mixed solution according to a ratio, reacting the DBM and ethanol mixed solution, the Phen and ethanol mixed solution and the rare earth ion and ethanol mixed solution, and filtering and drying to obtain fluorescent dye powder;
the specific steps of step S1 include:
s11: weighing raw materials including Eu 3+ Salt, DBM, Phen and absolute ethyl alcohol for later use; the ratio of the amounts of the substances in the step S11 is: eu (Eu) 3+ Weighing raw materials in a ratio of DBM to Phen to 1:3: 1;
s12: respectively dissolving DBM and Phen in absolute ethanol to obtain a mixed solution of DBM and ethanol and a mixed solution of Phen and ethanol, and adding Eu 3+ Dissolving salt in absolute ethyl alcohol to obtain an absolute ethyl alcohol mixed solution of rare earth ions;
s13: adding the DBM and ethanol mixed solution into an anhydrous ethanol solution of rare earth ions, placing the mixture into a three-neck flask, heating the mixture at 60-70 ℃, refluxing and stirring the mixture for reaction, dropwise adding the Phen and ethanol mixed solution into the three-neck flask by using a constant-pressure dropping funnel for continuous reaction, adjusting the pH value of the reaction solution, and then continuously refluxing and stirring the mixture for reaction until the reaction is finished; in the step S13, heating at 60-70 ℃, carrying out reflux stirring reaction for 0.5-1.5 h, dropwise adding a mixed solution of Phen and ethanol into a three-neck flask by using a constant-pressure dropping funnel, continuously reacting for 20-40 min, adjusting the pH value of the reaction solution to 6-7, and continuously heating, carrying out reflux stirring reaction for 3-5 h until the reaction is finished;
s14: transferring the reaction solution into a clean open container, standing at room temperature, cooling with ice water bath, vacuum filtering, and drying to obtain Eu (DBM) 3 Phen fluorescent dye powder; in the step S14, transferring the reaction solution into a clean open container, standing at room temperature for 24 hours, cooling with ice water bath for 20-40 min, performing suction filtration, and drying the filter cake at 60-70 ℃ to obtain Eu (DBM) 3 Phen fluorescent dye powder (i.e. fluorescent dye complex Eu (DBM)) 3 The Phen-fluorescence intensity is high, and the preparation cost is low);
preparation of S2 polystyrene microspheres: preparing polystyrene microspheres by a soap-free emulsion polymerization method to obtain white emulsion, namely polystyrene microsphere liquid (PSt);
the step S2 includes the following steps:
s21: adding 200mL of water and 10mL of refined styrene into a three-neck flask, introducing nitrogen, heating in a water bath, and stirring; the capacity of the three-mouth bottle in the step S21 is 500mL, and the three-mouth bottle is provided with a condensation pipe and a mechanical stirrer;
s22: heating in a water bath to 70-80 ℃, adding an initiator Ammonium Persulfate (APS) (dissolving 100mg of APS in 5mL of pure water), and continuously stirring for reacting for 20 hours to obtain a uniform white emulsion, namely polystyrene microsphere liquid (PSt);
s3 preparation of polystyrene color microspheres: mixing the polystyrene microsphere solution obtained in the step S2 with a color dye, emulsifying, and centrifuging to remove supernatant to obtain polystyrene color microspheres;
the step S3 includes the following steps:
s31: dispersing the polystyrene microsphere liquid obtained in the step S2 in a swelling medium; dissolving an oil-soluble yellow R dye in a swelling medium to obtain a dye swelling medium mixed solution;
in the step S31, 50 to 100 mul of polystyrene microsphere solution with the mass fraction of 10 percent is weighed and dispersed in a swelling medium (50 percent of acetone aqueous solution), and 5.2mg of oil-soluble yellow R dye is dissolved in the swelling medium (50 percent of acetone aqueous solution);
s32: mixing the microsphere solution obtained in the step S31 with a dye swelling medium mixed solution, and emulsifying, namely carrying out oscillation reaction for 1-3 h at room temperature; mixing the microsphere solution and the dye swelling medium mixed solution in the step S31 in the step S32, and then carrying out ultrasonic treatment for 0-15 min, preferably 5 min; carrying out ultrasonic treatment for 0-10 min every 1-2 h in the emulsification reaction; preferably, the ultrasonic treatment is carried out for 3min every 1 h;
s33: removing swelling medium (50% acetone water solution) by rotary evaporation at 40 deg.C, performing ultrasonic treatment, diluting the rest liquid gradually, centrifuging (at 16500rpm for 15min), and observing whether the centrifuged precipitate is complete and the color of the supernatant remains; discarding the supernatant, washing the lower-layer solid precipitate for multiple times by using pure water, removing the swelling medium solution, washing the lower-layer solid precipitate for multiple times by using the pure water until no color residue exists in the supernatant after centrifugation, removing the oil-soluble yellow R dye which does not enter the interior of the microsphere, and finally dissolving the obtained precipitate in 500 mu L of pure water to obtain PS colored microsphere solution; the ultrasonic treatment time in the step S33 is 1-15 min, preferably 5 min;
s4 preparation of polystyrene PS bicolor microspheres: mixing the fluorescent dye powder prepared in the step S1 with the polystyrene colorful microspheres prepared in the step S3, emulsifying, and centrifuging to remove supernatant to obtain polystyrene PS double-color microspheres;
the step S4 includes the following steps:
s41: dispersing the PS colored microsphere liquid (namely 5 mg-10 mg) obtained in the step S3 in 500 mu L swelling medium (50% acetone aqueous solution); eu (DBM) 3 Dissolving Phen fluorescent dye powder in a swelling medium (50% acetone aqueous solution) to obtain a fluorescent dye swelling medium mixed solution; in the step S41, 50 or 100 mul of polystyrene color microsphere liquid with the mass fraction of 10 percent is weighed and dispersed in a swelling medium;
s42: mixing the microsphere solution obtained in the step S541 and the mixed solution of the fluorescent dye swelling medium, and then emulsifying, namely carrying out oscillation reaction at room temperature, wherein in the step S42, the mixed solution of the microsphere solution obtained in the step S41 and the mixed solution of the dye swelling medium are mixed and then subjected to ultrasonic treatment, the ultrasonic treatment time is 0-15 min, preferably 5min, and the ultrasonic treatment is carried out for 0-10 min every 1-2 h in the emulsification reaction; preferably, the ultrasonic treatment is carried out for 3min every 1 h;
s43: removing swelling medium (50% acetone aqueous solution) by rotary evaporation at 40 deg.C, performing ultrasonic treatment, diluting the rest liquid, centrifuging, and observing whether the centrifuged precipitate is complete and fluorescence intensity of the supernatant; discarding supernatant, washing the lower layer solid precipitate with pure water for 3 times, removing swelling medium solution, washing with pure water for several times until the supernatant has no fluorescence after centrifugation, and removing Eu (DBM) not entering into microsphere 3 Phen, finally dissolving the obtained precipitate in 500 mu L of pure water to obtain PS bicolor microsphere solution; the time of ultrasonic treatment in the step S43 is 1-15 min, preferably 5 min.
Introducing a functional group for coupling an antigen or/and an antibody in the step S2 or step S3 or step S4. The functional group can be hydroxyl and is used for coupling biomolecules such as antigens and antibodies, so that the functional group can be further used for detection and analysis of biological samples and the detection sensitivity is improved.
Example 1: selecting an experiment of ultrasonic time in the step S42; the preparation method of the bicolor microsphere comprises the following steps:
preparation of S1 fluorescent dye: respectively preparing a DBM and ethanol mixed solution, a Phen and ethanol mixed solution and a rare earth ion and alcohol mixed solution according to a proportion, reacting the DBM and ethanol mixed solution, the Phen and ethanol mixed solution and the rare earth ion and alcohol mixed solution, and filtering and drying to obtain fluorescent dye powder;
the specific steps of step S1 include:
s11: weighing raw materials including EuCl 3 ·6H 2 O, DBM and Phen for standby; the ratio of the amounts of the substances in the step S11 is: weighing raw materials, namely Eu, DBM, Phen, 1, 3 and 1;
s12: respectively dissolving DBM and Phen in anhydrous ethanol to obtain DBM and ethanol mixed solution and Phen and ethanol mixed solution, and mixing EuCl 3 ·6H 2 Dissolving O in absolute ethyl alcohol to obtain an absolute ethyl alcohol mixed solution of rare earth ions; wherein 1.3455g of a first ligand DBM and 0.3604g of a second ligand Phen are respectively weighed and respectively dissolved in 10mL of absolute ethyl alcohol; 0.52g of the rare earth material EuCl was weighed out 3 ·6H 2 O, and dissolving in 20mL of absolute ethyl alcohol;
s13: adding a DBM and ethanol mixed solution into an anhydrous ethanol solution of rare earth ions, placing the mixture into a three-neck flask, heating the mixture at 65 ℃, refluxing and stirring the mixture for reaction for 1 hour, dropwise adding a Phen and ethanol mixed solution into the three-neck flask by using a constant-pressure dropping funnel, continuously reacting for 30 minutes, adjusting the pH value of a reaction solution to 6-7, continuously heating the mixture, refluxing and stirring the mixture for reaction for 4 hours until the reaction is finished;
s14: transferring the reaction solution into a clean open container, standing at room temperature for 24h, cooling with ice water bath for 30min, performing suction filtration by using a Buchner funnel, washing with absolute ethanol for 3-4 times in the suction filtration process to remove unreacted soluble impurities such as a second ligand Phen and the like in the product, and finally drying the filter cake obtained after suction filtration at 70 ℃ to obtain light yellow powder, namely Eu (DBM) 3 Phen fluorescent dye powder (using fluorescent dye)Complex Eu (DBM) 3 Phen-fluorescence intensity is high, and preparation cost is low);
preparation of S2 polystyrene microspheres: preparing polystyrene microspheres by a soap-free emulsion polymerization method to obtain white emulsion, namely polystyrene microsphere liquid (PSt); the step S2 includes the following steps:
s21: adding 200mL of water and 10mL of refined styrene into a three-mouth bottle with a condenser pipe and a mechanical stirrer, introducing nitrogen, heating in a water bath, and stirring; the volume of the three-mouth bottle in the step S21 is 500 mL;
s22: heating in water bath to 75 deg.C, adding initiator Ammonium Persulfate (APS) (dissolving 100mg ammonium persulfate in 5mL pure water), and stirring for 20 hr to obtain uniform white emulsion (polystyrene microsphere solution (PSt));
s3 preparation of polystyrene color microspheres: mixing and emulsifying the polystyrene microsphere liquid obtained in the step S2 by using a color dye, and centrifuging to remove supernatant liquid to obtain polystyrene color microspheres;
the step S3 includes the following steps:
s31: weighing 100 mu L of the polystyrene microsphere solution (10 mg of PS microsphere solution) with the mass fraction of 10 percent obtained in the step S2, and dispersing the polystyrene microsphere solution in a swelling medium (50 percent of acetone aqueous solution); 5.2mg of oil-soluble yellow R dye was dissolved in 80. mu.L of swelling medium (50% aqueous acetone);
s32: mixing the microsphere solution and the dye swelling medium mixed solution in the step S31, and emulsifying, namely carrying out oscillation reaction for 2h at room temperature; mixing the microsphere solution and the dye swelling medium mixed solution in the step S31 in the step S32, and then performing ultrasonic treatment for 5 min; carrying out ultrasonic treatment for 3min every 1h in the emulsification reaction;
s33: removing swelling medium (50% acetone aqueous solution) by rotary evaporation at 40 deg.C, performing ultrasonic treatment, diluting the rest liquid gradually, centrifuging, and observing whether the centrifuged precipitate is complete and fluorescence intensity of supernatant; discarding the supernatant, washing the lower-layer solid precipitate with pure water for 3 times, removing the swelling medium solution, washing with pure water for several times until no color residue exists in the supernatant after centrifugation, removing the oil-soluble yellow R dye which does not enter the interior of the microsphere, and finally dissolving the obtained precipitate in 500 mu L of pure water to obtain PS colored microsphere solution; the ultrasonic treatment time in the step S33 is 5 min;
s4 preparation of polystyrene PS bicolor microspheres: mixing the fluorescent dye powder prepared in the step S1 with the polystyrene colorful microspheres prepared in the step S3, emulsifying, and centrifuging to remove supernatant to obtain polystyrene PS double-color microspheres;
the step S4 includes the following steps:
s41: weighing 100 mul of the polystyrene colored microsphere liquid (namely 10mg) with the mass fraction of 10 percent obtained in the step S3, and dispersing the polystyrene colored microsphere liquid in 500 mul of swelling medium (50 percent of acetone aqueous solution); mixing 5.2mg Eu (DBM) 3 Dissolving Phen fluorescent dye powder in 80 μ L swelling medium (50% acetone water solution) to obtain fluorescent dye swelling medium mixed solution;
s42: mixing the microsphere solution and the mixed solution of the fluorescent dye swelling medium in the step S541, and then emulsifying, namely carrying out oscillation reaction at room temperature, wherein in the step S42, the microsphere solution and the mixed solution of the dye swelling medium in the step S41 are mixed and then are subjected to ultrasonic treatment for 0min, 1min, 5min and 15min respectively; carrying out ultrasonic treatment for 3min every 1h in the emulsification reaction;
s43: removing swelling medium (50% acetone aqueous solution) by rotary evaporation at 40 deg.C with a rotary evaporator, performing ultrasonic treatment for 5min, diluting the rest liquid gradually, centrifuging (the centrifugation speed is 16500rpm, the centrifugation time is 15min), and observing whether the centrifuged precipitate is complete and the fluorescence intensity of the supernatant; discarding the supernatant, washing the lower layer solid precipitate with pure water for 3 times, removing the swelling medium solution, washing with pure water for several times until the supernatant has no fluorescence after centrifugation, and removing Eu (DBM) which does not enter the interior of the microsphere 3 Phen, and finally dissolving the obtained precipitate in 500 mu L of pure water to obtain the PS bicolor microspheres.
Four PS bicolor microspheres obtained over different sonication times were passed through a particle size analyzer (Zetasizer Nano ZS), brand: MALVERN, model: Nano-ZS; detecting, wherein the detecting step comprises the following steps: 1. opening a particle size analyzer, and adjusting the detection temperature to 25 ℃; 2. adding 1200 mu L of pure water and 3 mu L of microspheres to be detected into the watch glass; 3. carrying out detection; the results are shown in Table 1.
TABLE 1 particle size detection results of PS bicolor microspheres obtained after different ultrasonic times
As can be seen from table 1, after swelling is finished, when ultrasound is not performed, the prepared microspheres partially agglomerate and have poor dispersibility, and the DPI is generally considered to be less than 0.1, so that the dispersibility is better, and the smaller the DPI is, the better the DPI is; when the ultrasonic treatment is carried out for 1min, the agglomeration phenomenon is improved a little; when the ultrasonic treatment is carried out for 5min or 15min, the prepared microspheres have good dispersibility and no agglomeration, the particle size of the microspheres is close to that of PS microspheres, and the ultrasonic treatment is comprehensively considered for 5min (the scanning electron microscope image of the microsphere is shown in figure 1).
Example 2: particle size analysis experiments of the PS double-color microspheres prepared by the invention (the preparation method of the specific embodiment is adopted, wherein the ultrasonic time is 5 min); adding 3 mu L of self-made bicolor microspheres into a watch glass, adding 1200 mu L of purified water, diluting and uniformly mixing, opening a particle size analyzer, adjusting the detection temperature to 25 ℃, and carrying out detection analysis.
The experimental instrumentation: particle size analyzer (Zetasizer Nano ZS), brand: MALVERN, model: Nano-ZS. The particle size detection results are shown in table 2 below, and the instrumental analysis results of the particle size of the microspheres are shown in fig. 2;
TABLE 2 particle size measurement results of PS dual-color microspheres
As shown in FIG. 2, the microspheres have an average particle size of 198nm, a dispersion coefficient of 0.002, good dispersibility, only 1 particle size peak and narrow particle size distribution.
In order to observe the particle size distribution of the microspheres more visually, the particle size of the microspheres is taken as the abscissa and the volume ratio is taken as the ordinate manually to observe the different particle size distribution conditions of the microspheres, as shown in fig. 3, as can be seen from fig. 3, the particle size distribution of the microspheres is narrow, and the comprehensive results show that the distribution of the microspheres is relatively uniform, and the microspheres serving as the color development marker have good stability and good use effect.
Example 3: the effect comparison experiment of the PS bicolor microspheres prepared by the invention and the commercially available monochromatic microspheres (colored microspheres and fluorescent microspheres);
first, the effect comparison experiment of the PS bicolor microsphere prepared by the invention and the fluorescent microsphere sold in the market. The method for preparing the PS double-color microspheres is a preparation method of a specific embodiment, wherein the ultrasonic time is 5min (a scanning electron microscope image of the PS double-color microspheres is shown as figure 1, and the figure 1 shows that the PS double-color microspheres prepared by the method have uniform size, good dispersibility and no agglomeration phenomenon); the commercially available monochromatic fluorescent microspheres are 200nm fluorescent microspheres with solid content of 1 percent; diluting 1 μ L of the extract with water in a ratio of 100000; both types of microspheres were tested in a time-resolved immunofluorescence analyzer (model: DR 6606; manufacturer: Darriy Biotechnology, Inc., Guangzhou, Inc.), and the results are shown in Table 3 below.
TABLE 3 detection results of PS bicolor microspheres prepared according to the present invention and Monochromatic-fluorescent microspheres
As can be seen from the comparative detection results in Table 3, the fluorescence intensity of the self-made two-color microspheres is higher than that of the commercially available single-color-fluorescent microspheres.
And secondly, effect comparison experiments of the PS bicolor microspheres prepared by the invention and commercial colored microspheres are carried out. The preparation method of the PS double-color microsphere prepared by the invention is a preparation method of a specific embodiment, wherein the ultrasonic time is 5min, and an immunochromatography detection effect comparison experiment of the PS double-color microsphere prepared by the invention and a commercial color microsphere (taking the detection of beta-human chorionic gonadotropin as an example, the microsphere is a carboxyl microsphere, and the preparation of the carboxyl microsphere is the prior art):
(1) labeling of the antibody: marking the two microspheres respectively, and adopting a classical EDC/NHS method to carry out coupling antibody marking; the method comprises the following specific steps: two centrifuge tubes were taken, and 100. mu.L of colored microspheres (solid content: 1%) were added, 880. mu.L of morpholine ethanesulfonic acid buffer (MES) (pH 6.0), 10. mu.L of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) at a mass concentration of 50mg/mL and 10. mu.L of NHS (N-hydroxysuccinimide) at a mass concentration of 30mg/mL, respectively, and activated in an incubator at 37 ℃ and 220rpm for 30 min; after activation, centrifuging at 15000rpm for 20min, removing supernatant, adding 1000 μ L PBS buffer solution to reset microspheres, adding 10 μ L beta-human chorionic gonadotropin protein solution with mass concentration of 2mg/mL, reacting in an incubator at 37 deg.C and 220rpm for 60min, centrifuging to remove supernatant, resetting with 1000 μ L PBS solution, and storing at 4 deg.C;
(2) respectively carrying out antibody marking on the PS bicolor microspheres and the purchased colored microspheres prepared by the method, and detecting by using an immunochromatography detection reagent strip produced by Nanjing Lanyu biology Limited company; observing the color development condition of the self-made bicolor microspheres and the commercially available monochromatic microspheres (yellow microspheres); respectively labeling the self-made double-color microspheres and the color microspheres with antibodies, then coating the microspheres on a combination pad of the reagent strip, and simultaneously coating the chicken IgY antibodies (coating concentration: 0.5mg/mL) on the combination pad;
the method specifically comprises the following steps:
1) assembling the reagent strip: cutting the NC membrane, the sample pad, the absorbent paper and the PVC base plate into 3mm, sequentially sticking the NC membrane, the sample pad and the absorbent paper on the PVC base plate in sequence as shown in figure 4, and storing in a 4 ℃ dry place;
2) 2 mu L of 2mg/mL beta-human chorionic gonadotropin antibody is absorbed by a 10 mu L pipette and is dripped on one end of an NC membrane close to a sample pad to form a detection line, and a quality control line (goat anti-chicken IgY antibody, 1mg/mL) is arranged at the same time;
3) the immunochromatography detection reagent cards prepared from the self-made microspheres and the purchased colored microspheres are respectively detected, namely, purified water is taken as a sample and added into the reagent card, and the color development conditions of the reagent strips prepared from the self-made bicolor microspheres and the commercially available colored microspheres are observed, so that the result is shown in figure 5, and the detection results of the self-made bicolor microspheres and the commercially available colored microspheres can be seen from figure 5, and the color development degrees of the self-made bicolor microspheres and the commercially available colored microspheres are equivalent.
The contrast experiment results of the self-made double-color microspheres, the color microspheres and the fluorescent microspheres show that the fluorescence intensity of the self-made double-color microspheres is stronger than that of the purchased fluorescent microspheres, and the color depth is close to that of the purchased colored microspheres in the aspect of color development; the bicolor microspheres prepared by the method can be applied to microsphere markers in immunochromatography detection and analysis, and the detection result is reliable.
The above-mentioned embodiments, objects, technical solutions and advantages of the present invention are further described in detail, it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention, and are not intended to limit the present invention, and any modifications, equivalent substitutions, improvements and the like, such as changes in shape or material of some components, may be made within the spirit and principle of the present invention; are intended to be included within the scope of the present invention.
Claims (11)
1. A preparation method of bicolor microspheres is characterized by comprising the following steps:
preparation of S1 fluorescent dye: respectively preparing a DBM and ethanol mixed solution, a Phen and ethanol mixed solution and a rare earth ion and ethanol mixed solution according to a certain proportion, reacting the DBM and ethanol mixed solution and the Phen and ethanol mixed solution with the rare earth ion and ethanol mixed solution, filtering, drying to obtain fluorescent dye powder;
preparation of S2 polystyrene microspheres: preparing polystyrene microspheres by a soap-free emulsion polymerization method to obtain white emulsion, namely polystyrene microsphere liquid;
s3 preparation of polystyrene color microspheres: mixing and emulsifying the polystyrene microsphere liquid obtained in the step S2 by using a color dye, and centrifuging to remove supernatant liquid to obtain polystyrene color microspheres;
s4 preparation of polystyrene PS bicolor microspheres: and (3) mixing the fluorescent dye powder prepared in the step (S1) with the polystyrene colorful microspheres prepared in the step (S3), emulsifying, and centrifuging to remove supernatant liquid to obtain the polystyrene PS double-color microspheres.
2. The method for preparing two-color microspheres according to claim 1, wherein the specific step of step S1 comprises:
s11: weighing raw materials including Eu 3+ Salt, DBM and Phen for standby;
s12: respectively dissolving DBM and Phen in absolute ethyl alcohol to obtain a mixed solution of DBM and ethyl alcohol and a mixed solution of Phen and ethyl alcohol; eu is mixed 3+ Dissolving salt in absolute ethyl alcohol to obtain an absolute ethyl alcohol mixed solution of rare earth ions;
s13: adding the DBM and ethanol mixed solution into an anhydrous ethanol solution of rare earth ions, placing the mixture into a three-neck flask, heating the mixture at 60-70 ℃, carrying out reflux stirring reaction, dropwise adding the Phen and ethanol mixed solution into the three-neck flask by using a constant-pressure dropping funnel, continuously reacting, adjusting the pH value of the reaction solution, and continuously carrying out reflux stirring reaction until the reaction is finished;
s14: transferring the reaction solution into a clean open container, standing at room temperature, cooling with ice water bath, vacuum filtering, and drying to obtain Eu (DBM) 3 Phen fluorescent dye powder.
3. The method for preparing the bichromal microsphere according to claim 2, wherein the step S2 comprises the following steps:
s21: adding 200mL of water and 10mL of refined styrene into a three-neck flask, introducing nitrogen, heating in a water bath, and stirring;
s22: and (3) when the temperature of the water bath is raised to 70-80 ℃, adding initiator ammonium persulfate, and continuously stirring for reaction to obtain uniform white emulsion, namely the polystyrene microsphere liquid.
4. The method for preparing the bicolor microsphere according to claim 3, wherein the step S3 comprises the following specific steps:
s31: dispersing the polystyrene microsphere liquid obtained in the step S2 in a swelling medium; dissolving an oil-soluble yellow R dye in a swelling medium to obtain a dye swelling medium mixed solution;
s32: mixing the microsphere solution obtained in the step S31 with a mixed solution of a dye swelling medium, and emulsifying, namely carrying out oscillation reaction for 1-3 h at room temperature;
s33: and (3) removing the swelling medium by rotary evaporation, performing ultrasonic treatment, gradually diluting the residual liquid, centrifuging, removing the supernatant, washing the lower-layer solid precipitate for multiple times by using pure water, and finally dissolving the obtained precipitate in the pure water to obtain the PS colored microsphere liquid.
5. The method for preparing the bichromal microsphere according to claim 4, wherein the step S4 comprises the following steps:
s41: dispersing the PS colored microsphere liquid obtained in the step S3 in a swelling medium; eu (DBM) 3 Dissolving Phen fluorescent dye powder in a swelling medium to obtain a fluorescent dye swelling medium mixed solution;
s42: mixing the microsphere solution obtained in the step S541 and the mixed solution of the fluorescent dye swelling medium, and emulsifying, namely carrying out oscillation reaction at room temperature;
s43: and (3) removing the swelling medium by rotary evaporation, performing ultrasonic treatment, gradually diluting the residual liquid, centrifuging, removing the supernatant, washing the lower-layer solid precipitate for multiple times by using pure water, and finally dissolving the obtained precipitate in the pure water to obtain the PS double-color microsphere liquid.
6. The method for preparing two-color microspheres according to claim 2, wherein the ratio of the amounts of the substances in step S11 is: eu (Eu) 3+ DBM, Phen 1:3: 1; in the step S13, firstly heating at 60-70 ℃, carrying out reflux stirring reaction for 0.5-1.5 h, then dropwise adding the mixed solution of Phen and ethanol into a three-neck flask by using a constant-pressure dropping funnel, continuously reacting for 20-40 min, adjusting the pH value of the reaction solution to 6-7, and then continuously heating, carrying out reflux stirring reaction for 3-5 h until the reaction is finished; in the step S14, transferring the reaction solution into a clean open container, standing at room temperature for 24 hours, cooling with ice water bath for 20-40 min, performing suction filtration, and drying the filter cake at 60-70 ℃ to obtain Eu (DBM) 3 Phen fluorescent dye powder.
7. The method for preparing bichromal microballoons of claim 3, wherein the capacity of the three-necked bottle in step S21 is 500mL, and the three-necked bottle is provided with a condenser tube and a mechanical stirrer.
8. The preparation method of two-color microspheres according to claim 4, wherein 50 to 100 μ L of polystyrene microsphere solution with a mass fraction of 10% is weighed in the step S31 and dispersed in a swelling medium, and 4 to 5.5mg of oil-soluble yellow R dye is dissolved in 70 to 85 μ L of the swelling medium; mixing the microsphere solution and the dye swelling medium mixed solution in the step S31 in the step S32, and then carrying out ultrasonic treatment for 0-15 min, wherein ultrasonic treatment is carried out for 0-10 min every 1-2 h in the emulsification reaction; the ultrasonic treatment time in the step S33 is 1-15 min.
9. The method for preparing two-color microspheres according to claim 5, wherein 50 to 100 μ L of a polystyrene colored microsphere solution with a mass fraction of 10% is weighed in a swelling medium in step S41; 4 to 5.5mg of Eu (DBM) 3 Dissolving Phen fluorescent dye powder in 70-85 mu L of swelling medium; in the step S42, the microsphere solution and the dye swelling medium mixed solution in the step S41 are mixed and then subjected to ultrasonic treatment, wherein the ultrasonic treatment time is 0-15 min, and ultrasonic treatment is performed for 0-10 min every 1-2 h in an emulsification reaction; the ultrasonic treatment time in the step S43 is 1-15 min.
10. The method for preparing two-color microspheres according to claim 5, wherein a functional group is introduced for coupling an antigen or/and an antibody in step S2 or step S3 or step S4.
11. The application of the bicolor microspheres is characterized in that the bicolor microspheres are applied to a microsphere marker in immunochromatography detection.
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