CN108359059B - Preparation method and application of fluorescent microspheres - Google Patents
Preparation method and application of fluorescent microspheres Download PDFInfo
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- 239000004005 microsphere Substances 0.000 title claims abstract description 84
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 239000000243 solution Substances 0.000 claims description 178
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 57
- 239000007864 aqueous solution Substances 0.000 claims description 47
- 239000002096 quantum dot Substances 0.000 claims description 43
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 claims description 38
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 claims description 20
- 229960000907 methylthioninium chloride Drugs 0.000 claims description 20
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 claims description 19
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims description 19
- DBCAQXHNJOFNGC-UHFFFAOYSA-N 4-bromo-1,1,1-trifluorobutane Chemical compound FC(F)(F)CCCBr DBCAQXHNJOFNGC-UHFFFAOYSA-N 0.000 claims description 19
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 claims description 19
- STVZJERGLQHEKB-UHFFFAOYSA-N ethylene glycol dimethacrylate Substances CC(=C)C(=O)OCCOC(=O)C(C)=C STVZJERGLQHEKB-UHFFFAOYSA-N 0.000 claims description 19
- OMPJBNCRMGITSC-UHFFFAOYSA-N Benzoylperoxide Chemical compound C=1C=CC=CC=1C(=O)OOC(=O)C1=CC=CC=C1 OMPJBNCRMGITSC-UHFFFAOYSA-N 0.000 claims description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 18
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 claims description 18
- 235000019400 benzoyl peroxide Nutrition 0.000 claims description 18
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 18
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 18
- 229940069328 povidone Drugs 0.000 claims description 18
- 238000003756 stirring Methods 0.000 claims description 18
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 18
- 239000012498 ultrapure water Substances 0.000 claims description 18
- 238000010790 dilution Methods 0.000 claims description 10
- 239000012895 dilution Substances 0.000 claims description 10
- 239000004793 Polystyrene Substances 0.000 claims description 9
- 230000005484 gravity Effects 0.000 claims description 9
- 229920002223 polystyrene Polymers 0.000 claims description 9
- 238000004321 preservation Methods 0.000 claims description 9
- 238000012216 screening Methods 0.000 claims description 9
- 238000001132 ultrasonic dispersion Methods 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 5
- 238000000684 flow cytometry Methods 0.000 claims description 3
- 239000000463 material Substances 0.000 abstract description 2
- 230000009286 beneficial effect Effects 0.000 description 8
- 238000001514 detection method Methods 0.000 description 5
- 206010042674 Swelling Diseases 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 230000008961 swelling Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000000295 emission spectrum Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 1
- CHIHQLCVLOXUJW-UHFFFAOYSA-N benzoic anhydride Chemical compound C=1C=CC=CC=1C(=O)OC(=O)C1=CC=CC=C1 CHIHQLCVLOXUJW-UHFFFAOYSA-N 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000000695 excitation spectrum Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000002522 swelling effect Effects 0.000 description 1
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- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F257/00—Macromolecular compounds obtained by polymerising monomers on to polymers of aromatic monomers as defined in group C08F12/00
- C08F257/02—Macromolecular compounds obtained by polymerising monomers on to polymers of aromatic monomers as defined in group C08F12/00 on to polymers of styrene or alkyl-substituted styrenes
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- C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
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- C08J2325/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an aromatic carbocyclic ring; Derivatives of such polymers
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Abstract
The invention discloses a preparation method and application of fluorescent microspheres, belonging to the field of biomedical materials.
Description
Technical Field
The invention belongs to the field of biomedical materials, and particularly relates to a preparation method and application of fluorescent microspheres.
Background
Compared with the traditional fluorescent dye, the quantum dot has the advantages of good light stability, wide and continuous excitation spectrum, narrow and symmetrical emission spectrum, larger Stokes shift, good biocompatibility and longer fluorescence life. More remarkably, in a certain range, the quantum dots can also realize the regulation and control of the emission spectrum of the quantum dots by adjusting the size of the quantum dots; the solubility can be adjusted according to the surface modification. The quantum dots have wide application in the field of life science due to the characteristics, for example, the quantum dot fluorescent microspheres replace the traditional organic dye fluorescent microspheres to be used for liquid phase chips, so that the quantum dot fluorescent microspheres have more excellent performance and more convenient storage conditions.
The existing method for preparing the quantum dot fluorescent microspheres is basically finished in two steps, namely, porous microspheres are prepared firstly, and then quantum dots are adsorbed by a physical adsorption mode to obtain the fluorescent microspheres. However, the quantum dots are mainly concentrated on the surface or shallow layer of the microsphere, so that the fluorescent microsphere has unstable fluorescence characteristics and non-uniform fluorescence intensity. These defects also make the quantum dot fluorescent microsphere have certain obstacles in practical application.
Disclosure of Invention
In order to solve the above problems, an object of the present invention is to provide a method for preparing a fluorescent microsphere containing quantum dots, which is added in a swelling polymerization stage to obtain quantum dots having uniform and stable fluorescence intensity.
In order to achieve the purpose, the technical scheme of the invention is as follows: a preparation method of fluorescent microspheres comprises the following steps:
1) adding the seed microspheres into 0.25 wt% SDS aqueous solution, and performing ultrasonic dispersion to obtain solution A with the concentration of 0.001-0.025g/m L;
2) ultrasonically dispersing cyclohexane in 0.25 wt% SDS water solution to obtain solution B with concentration of 0.001-0.025g/m L;
3) dropwise adding the solution B obtained in the step 2) into the solution A to obtain a solution C, wherein the mass ratio of the seed microspheres to cyclohexane in the solution C is 1-5: 1-5, stirring the solution C at room temperature to swell for 4-8 h;
4) and (2) sequentially adding dibenzoyl peroxide, styrene, ethylene glycol dimethacrylate, toluene and acrylic acid into the solution C, wherein the mass ratio of the dibenzoyl peroxide, the styrene, the ethylene glycol dimethacrylate, the toluene, the acrylic acid and the seed microspheres is as follows: 1-5:100-500:100-500: 100-500:5-25: 1-5;
5) adding 0.25 wt% SDS aqueous solution into the solution obtained in the step (4) for dilution, wherein the volume of the added SDS aqueous solution is 0.4-2 times of the volume of the solution obtained in the step (4), then adding 1/10000-1/100 quantum dot solution into the solution after dilution, and stirring for 6-12h after the quantum dot solution is added;
6) carrying out oil bath heat preservation on the solution obtained in the step 5) at 75-85 ℃, and sequentially adding povidone, a methylene blue aqueous solution with the concentration of 6-9mg/m L and ultrapure water for reacting for 6-18h, wherein the mass ratio of the povidone to the seed microspheres in the solution A is 20-100:1-5, and the adding amounts of the methylene blue aqueous solution and the ultrapure water are respectively 1/700-1/6 and 5/28-5/12 of the total volume of the solution obtained in the step 5);
7) washing the solution obtained in the step 6) with 10 vol% ethanol, and performing gravity screening to obtain the carboxylated porous polystyrene quantum dot fluorescent microspheres with uniform size.
The beneficial effects of the above technical scheme are that: by adding quantum dot solution to mix in the swelling stage of the microspheres, the quantum dot solution is easier to coat into the microspheres and is uniformly dispersed in the microspheres, and finally the fluorescence intensity of each microsphere is uniform and stable, which is beneficial to improving the detection accuracy.
The concentration of the solution A in the step 1) in the technical scheme is 0.005-0.02g/m L.
The beneficial effects of the above technical scheme are that: the concentration is convenient to prepare and use, and the swelling is facilitated.
The concentration of the B solution in the step 2) in the technical scheme is 0.005-0.02g/m L.
The beneficial effects of the above technical scheme are that: the concentration is convenient to prepare and use, and the swelling is facilitated.
In the above technical scheme, the mass ratio of the seed microspheres to cyclohexane in the solution C in the step 3) is 2: 1-5.
The beneficial effects of the above technical scheme are that: moderate proportion and good swelling effect.
In the above technical scheme, the mass ratio of dibenzoyl oxide, styrene, ethylene glycol dimethacrylate, toluene, acrylic acid and seed microspheres in step 4) is as follows: 3:200-400:200-400: 200-400:10-20:2-4.
The beneficial effects of the above technical scheme are that: the proportion is moderate, and the size and the shape of the microsphere can be regulated and controlled.
The quantum dot solution added in the step 5) in the technical scheme is 1/1000-1/100 of the total volume of the diluted solution.
The beneficial effects of the above technical scheme are that: easy preparation and convenient use.
The concentration of the methylene blue aqueous solution in the step 6) in the technical scheme is 7.5mg/m L.
The technical scheme has the beneficial effects of moderate concentration and convenient use.
The invention also aims to provide the application of the fluorescent microspheres prepared by the preparation method in flow cytometry detection.
Drawings
FIG. 1 is a flow chart illustrating a method for preparing fluorescent microspheres according to an embodiment of the present invention.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.
The seed microspheres in the following examples were purchased from Wuhan Huake Microscience and technology, Inc., with a product model of PS-M-10035 and a product specification of 5% (w/v).
Example 1
The embodiment provides a preparation method of fluorescent microspheres, which is characterized by comprising the following steps:
1) adding the seed microspheres into 0.25 wt% SDS aqueous solution, and performing ultrasonic dispersion to obtain solution A with the concentration of 0.001g/m L;
2) ultrasonically dispersing cyclohexane in 0.25 wt% SDS water solution to obtain solution B with concentration of 0.001g/m L;
3) dropwise adding the solution B obtained in the step 2) into the solution A to obtain a solution C, wherein the mass ratio of the seed microspheres to cyclohexane in the solution C is 1:1, stirring the solution C at room temperature to swell for 4-8 h;
4) and (2) sequentially adding dibenzoyl peroxide, styrene, ethylene glycol dimethacrylate, toluene and acrylic acid into the solution C, wherein the mass ratio of the dibenzoyl peroxide, the styrene, the ethylene glycol dimethacrylate, the toluene, the acrylic acid and the seed microspheres is as follows: 1:100:100: 100:5: 1;
5) adding an SDS aqueous solution with the concentration of 0.25 wt% into the solution obtained in the step (4) for dilution, wherein the volume of the added SDS aqueous solution is 0.4 times of that of the solution obtained in the step 4), then adding 1/10000 quantum dot solutions of the total volume of the diluted solution into the solution, and stirring for 6 hours after the quantum dot solution is added;
6) carrying out oil bath heat preservation on the solution obtained in the step 5) at 75 ℃, and sequentially adding povidone, a methylene blue aqueous solution with the concentration of 6mg/m L and ultrapure water for reacting for 6h, wherein the mass ratio of the povidone to the seed microspheres in the solution A is 20:1, and the addition amounts of the methylene blue aqueous solution and the ultrapure water are respectively 1/700 and 5/28 of the total volume of the solution obtained in the step 5);
7) washing the solution obtained in the step 6) with 10 vol% ethanol, and performing gravity screening to obtain the carboxylated porous polystyrene quantum dot fluorescent microspheres with uniform size.
Example 2
The embodiment provides a preparation method of fluorescent microspheres, which comprises the following steps:
1) adding the seed microspheres into 0.25 wt% SDS aqueous solution, and performing ultrasonic dispersion to obtain solution A with the concentration of 0.01g/m L;
2) ultrasonically dispersing cyclohexane in 0.25 wt% SDS water solution to obtain solution B with concentration of 0.01g/m L;
3) dropwise adding the solution B obtained in the step 2) into the solution A to obtain a solution C, wherein the mass ratio of the seed microspheres to cyclohexane in the solution C is 2: 3, stirring the solution C at room temperature to swell for 6 hours;
4) and (2) sequentially adding dibenzoyl peroxide, styrene, ethylene glycol dimethacrylate, toluene and acrylic acid into the solution C, wherein the mass ratio of the dibenzoyl peroxide, the styrene, the ethylene glycol dimethacrylate, the toluene, the acrylic acid and the seed microspheres is as follows: 3:300:300: 300:15: 2;
5) adding an SDS aqueous solution with the concentration of 0.25 wt% into the solution obtained in the step (4) for dilution, wherein the volume of the added SDS aqueous solution is 1.2 times of that of the solution obtained in the step 4), then adding 1/500 quantum dot solutions of the total volume of the diluted solution into the solution, and stirring the solution for 9 hours after the quantum dot solution is added;
6) carrying out oil bath heat preservation on the solution obtained in the step 5) at the temperature of 80 ℃, and sequentially adding povidone, a methylene blue aqueous solution with the concentration of 7.5mg/m L and ultrapure water for reaction for 12 hours, wherein the mass ratio of the povidone to the seed microspheres in the solution A is 20:1, and the addition amounts of the methylene blue aqueous solution and the ultrapure water are respectively 1/50 and 1/4 of the total volume of the solution obtained in the step 5);
7) washing the solution obtained in the step 6) with 10 vol% ethanol, and performing gravity screening to obtain the carboxylated porous polystyrene quantum dot fluorescent microspheres with uniform size.
Example 3
The embodiment provides a preparation method of fluorescent microspheres, which comprises the following steps:
1) adding the seed microspheres into 0.25 wt% SDS aqueous solution, and performing ultrasonic dispersion to obtain solution A with the concentration of 0.025g/m L;
2) ultrasonically dispersing cyclohexane in 0.25 wt% SDS water solution to obtain solution B with concentration of 0.025g/m L;
3) dropwise adding the solution B obtained in the step 2) into the solution A to obtain a solution C, wherein the mass ratio of the seed microspheres to cyclohexane in the solution C is 1: 5, stirring the solution C at room temperature to swell for 8 hours;
4) and (2) sequentially adding dibenzoyl peroxide, styrene, ethylene glycol dimethacrylate, toluene and acrylic acid into the solution C, wherein the mass ratio of the dibenzoyl peroxide, the styrene, the ethylene glycol dimethacrylate, the toluene, the acrylic acid and the seed microspheres is as follows: 1:20:20: 20:1: 1;
5) adding an SDS aqueous solution with the concentration of 0.25 wt% into the solution obtained in the step (4) for dilution, wherein the volume of the added SDS aqueous solution is 2 times of that of the solution obtained in the step 4), then adding 1/100 quantum dot solutions of the total volume of the diluted solution into the solution, and stirring the solution for 12 hours after the quantum dot solution is added;
6) carrying out oil bath heat preservation on the solution in the step 5) at 85 ℃, and sequentially adding povidone, a methylene blue aqueous solution with the concentration of 9mg/m L and ultrapure water for reacting for 18h, wherein the mass ratio of the povidone to the seed microspheres in the solution A is 100:1, and the addition amounts of the methylene blue aqueous solution and the ultrapure water are respectively 1/6 and 5/12 of the total volume of the solution obtained in the step 5);
7) washing the solution obtained in the step 6) with 10 vol% ethanol, and performing gravity screening to obtain the carboxylated porous polystyrene quantum dot fluorescent microspheres with uniform size.
Example 4
The embodiment provides a preparation method of fluorescent microspheres, which comprises the following steps:
1) adding the seed microspheres into 0.25 wt% SDS aqueous solution, and performing ultrasonic dispersion to obtain solution A with the concentration of 0.005g/m L;
2) ultrasonically dispersing cyclohexane in 0.25 wt% SDS water solution to obtain solution B with concentration of 0.005g/m L;
3) dropwise adding the solution B obtained in the step 2) into the solution A to obtain a solution C, wherein the mass ratio of the seed microspheres to cyclohexane in the solution C is 2: 1, stirring the solution C at room temperature to swell for 4-8 h;
4) and (2) sequentially adding dibenzoyl peroxide, styrene, ethylene glycol dimethacrylate, toluene and acrylic acid into the solution C, wherein the mass ratio of the dibenzoyl peroxide, the styrene, the ethylene glycol dimethacrylate, the toluene, the acrylic acid and the seed microspheres is as follows: 3:200:200: 200:10: 2;
5) adding an SDS aqueous solution with the concentration of 0.25 wt% into the solution obtained in the step (4) for dilution, wherein the volume of the added SDS aqueous solution is 1 time of that of the solution obtained in the step 4), then adding 1/1000 quantum dot solutions of the total volume of the diluted solution into the solution, and stirring for 8 hours after the quantum dot solutions are added;
6) carrying out oil bath heat preservation on the solution in the step 5) at 83 ℃, and sequentially adding povidone, a methylene blue aqueous solution with the concentration of 7.5mg/m L and ultrapure water for reaction for 13h, wherein the mass ratio of the povidone to the seed microspheres in the solution A is 100:1, and the addition amounts of the methylene blue aqueous solution and the ultrapure water are respectively 1/100 and 1/5 of the total volume of the solution obtained in the step 5);
7) washing the solution obtained in the step 6) with 10 vol% ethanol, and performing gravity screening to obtain the carboxylated porous polystyrene quantum dot fluorescent microspheres with uniform size.
Example 5
The embodiment provides a preparation method of fluorescent microspheres, which comprises the following steps:
1) adding the seed microspheres into 0.25 wt% SDS aqueous solution, and performing ultrasonic dispersion to obtain solution A with the concentration of 0.02g/m L;
2) ultrasonically dispersing cyclohexane in 0.25 wt% SDS water solution to obtain solution B with concentration of 0.02g/m L;
3) dropwise adding the solution B obtained in the step 2) into the solution A to obtain a solution C, wherein the mass ratio of the seed microspheres to cyclohexane in the solution C is 2: 5, stirring the solution C at room temperature to swell for 7 hours;
4) and (2) sequentially adding dibenzoyl peroxide, styrene, ethylene glycol dimethacrylate, toluene and acrylic acid into the solution C, wherein the mass ratio of the dibenzoyl peroxide, the styrene, the ethylene glycol dimethacrylate, the toluene, the acrylic acid and the seed microspheres is as follows: 3:400:400: 400:20: 4;
5) adding an SDS aqueous solution with the concentration of 0.25 wt% into the solution obtained in the step (4) for dilution, wherein the volume of the added SDS aqueous solution is 0.4-2 times of the volume of the solution obtained in the step 4), then adding 1/100 quantum dot solutions of the total volume of the diluted solution, and stirring for 12 hours after the quantum dot solution is added;
6) carrying out oil bath heat preservation on the solution in the step 5) at 78 ℃, and sequentially adding povidone, a methylene blue aqueous solution with the concentration of 7.5mg/m L and ultrapure water for reacting for 16h, wherein the mass ratio of the povidone to the seed microspheres in the solution A is 50:1, and the addition amounts of the methylene blue aqueous solution and the ultrapure water are respectively 1/12 and 5/12 of the total volume of the solution obtained in the step 5);
7) washing the solution obtained in the step 6) with 10 vol% ethanol, and performing gravity screening to obtain the carboxylated porous polystyrene quantum dot fluorescent microspheres with uniform size.
Comparative example 1
The embodiment provides a preparation method of fluorescent microspheres, which comprises the following steps:
1) adding the seed microspheres into 0.25 wt% SDS aqueous solution, and performing ultrasonic dispersion to obtain solution A with the concentration of 0.005g/m L;
2) ultrasonically dispersing cyclohexane in 0.25 wt% SDS water solution to obtain solution B with concentration of 0.005g/m L;
3) dropwise adding the solution B obtained in the step 2) into the solution A to obtain a solution C, wherein the mass ratio of the seed microspheres to cyclohexane in the solution C is 6: 1, stirring the solution C at room temperature to swell for 4 hours;
4) and (2) sequentially adding dibenzoyl peroxide, styrene, ethylene glycol dimethacrylate, toluene and acrylic acid into the solution C, wherein the mass ratio of the dibenzoyl peroxide, the styrene, the ethylene glycol dimethacrylate, the toluene, the acrylic acid and the seed microspheres is as follows: 1:10:10: 10:3: 1;
5) adding an SDS aqueous solution with the concentration of 0.25 wt% into the solution obtained in the step (4) for dilution, wherein the volume of the added SDS aqueous solution is 0.1 time of that of the solution obtained in the step 4), then adding 1/50 quantum dot solutions of the total volume of the diluted solution into the solution, and stirring the solution for 12 hours after the quantum dot solution is added;
6) carrying out oil bath heat preservation on the solution in the step 5) at 70 ℃, and sequentially adding povidone, a methylene blue aqueous solution with the concentration of 5mg/m L and ultrapure water for reacting for 18h, wherein the mass ratio of the povidone to the seed microspheres in the solution A is 20:6, and the addition amounts of the methylene blue aqueous solution and the ultrapure water are respectively 1/800 and 1/6 of the total volume of the solution obtained in the step 5);
7) washing the solution obtained in the step 6) with 5 vol% ethanol and carrying out gravity screening to obtain the carboxylated porous polystyrene quantum dot fluorescent microspheres with uniform size.
Comparative example 2
The embodiment provides a preparation method of fluorescent microspheres, which is characterized by comprising the following steps:
1) adding the seed microspheres into 0.25 wt% SDS aqueous solution, and performing ultrasonic dispersion to obtain solution A with the concentration of 0.03g/m L;
2) ultrasonically dispersing cyclohexane in 0.25 wt% SDS water solution to obtain solution B with concentration of 0.03g/m L;
3) dropwise adding the solution B obtained in the step 2) into the solution A to obtain a solution C, wherein the mass ratio of the seed microspheres to cyclohexane in the solution C is 1: stirring the solution C at room temperature to swell for 8 hours;
4) and (2) sequentially adding dibenzoyl peroxide, styrene, ethylene glycol dimethacrylate, toluene and acrylic acid into the solution C, wherein the mass ratio of the dibenzoyl peroxide, the styrene, the ethylene glycol dimethacrylate, the toluene, the acrylic acid and the seed microspheres is as follows: 1:600:600: 60:30: 6;
5) adding an SDS aqueous solution with the concentration of 0.25 wt% into the solution obtained in the step (4) for dilution, wherein the volume of the added SDS aqueous solution is 3 times of that of the solution obtained in the step 4), then adding 1/10 quantum dot solutions of the total volume of the diluted solution into the solution, and stirring the solution for 12 hours after the quantum dot solution is added;
6) carrying out oil bath heat preservation on the solution obtained in the step 5) at 90 ℃, and sequentially adding povidone, a methylene blue aqueous solution with the concentration of 6-9mg/m L and ultrapure water for reacting for 6-18h, wherein the mass ratio of the povidone to the seed microspheres in the solution A is 110:1, and the addition amounts of the methylene blue aqueous solution and the ultrapure water are respectively 1/5 and 1/2 of the total volume of the solution obtained in the step 5);
7) washing the solution obtained in the step 6) with 10 vol% ethanol, and performing gravity screening to obtain the carboxylated porous polystyrene quantum dot fluorescent microspheres with uniform size.
Analysis of results
Through carrying out loss cell detection on the fluorescent microspheres obtained by seven groups of experiments in total in examples 1-5 and comparative examples 1-2, the fluorescent microspheres corresponding to examples 1-5 are found to have strong and stable fluorescence intensity uniformity and can be applied to loss cell detection; the fluorescent microspheres corresponding to the comparative examples 1-2 have uneven fluorescence intensity and are unstable, so that the fluorescent microspheres are not suitable for flow cytometry detection.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (3)
1. The preparation method of the fluorescent microsphere is characterized by comprising the following steps:
1) adding the seed microspheres into 0.25 wt% SDS aqueous solution, and performing ultrasonic dispersion to obtain solution A with the concentration of 0.005-0.02g/m L;
2) ultrasonically dispersing cyclohexane in 0.25 wt% SDS water solution to obtain solution B with concentration of 0.02-0.025g/m L;
3) dropwise adding the solution B obtained in the step 2) into the solution A to obtain a solution C, wherein the mass ratio of the seed microspheres to cyclohexane in the solution C is 2: 3-10, stirring the solution C at room temperature to swell for 4-8 h;
4) and (2) sequentially adding dibenzoyl peroxide, styrene, ethylene glycol dimethacrylate, toluene and acrylic acid into the solution C, wherein the mass ratio of the dibenzoyl peroxide, the styrene, the ethylene glycol dimethacrylate, the toluene, the acrylic acid and the seed microspheres is as follows: 3:200-400:200-400: 200-400:10-20: 2-4;
5) adding 0.25 wt% SDS aqueous solution into the solution obtained in the step (4) for dilution, wherein the volume of the added SDS aqueous solution is 0.4-2 times of the volume of the solution obtained in the step 4), then adding 1/1000-1/100 quantum dot solution into the solution, and stirring for 6-12h after the quantum dot solution is added;
6) carrying out oil bath heat preservation on the solution obtained in the step 5) at 75-85 ℃, and sequentially adding povidone, a methylene blue aqueous solution with the concentration of 6-9mg/m L and ultrapure water for reacting for 6-18h, wherein the mass ratio of the povidone to the seed microspheres in the solution A is 20-100:1-5, and the adding amounts of the methylene blue aqueous solution and the ultrapure water are respectively 1/700-1/6 and 5/28-5/12 of the total volume obtained in the step 5);
7) washing the solution obtained in the step 6) with 10 vol% ethanol, and performing gravity screening to obtain the carboxylated porous polystyrene quantum dot fluorescent microspheres with uniform size.
2. The method for preparing fluorescent microspheres of claim 1, wherein the concentration of the aqueous solution of methylene blue in step 6) is 7.5mg/m L.
3. Use of a fluorescent microsphere prepared by the method of claim 1 or 2 in flow cytometry.
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