CN105131156A - Preparation method of surface aminated polystyrene microspheres - Google Patents
Preparation method of surface aminated polystyrene microspheres Download PDFInfo
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- CN105131156A CN105131156A CN201510521756.5A CN201510521756A CN105131156A CN 105131156 A CN105131156 A CN 105131156A CN 201510521756 A CN201510521756 A CN 201510521756A CN 105131156 A CN105131156 A CN 105131156A
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Abstract
The invention belongs to the technical field of preparation of polymer microsphere materials, and concretely relates to a preparation method of surface aminated polystyrene microspheres. The method comprises the following steps: preparing polystyrene microspheres, aminating the polystyrene microspheres, and carrying out streptavidin treatment. The method has the advantages of mild conditions, high yield, simplicity, and suitableness for large-scale popularization application, and can meet gene sequencing needs.
Description
Technical field
The invention belongs to polymer microballoon technical field of material, be specifically related to a kind of preparation method of surface amination polystyrene microsphere.
Background technology
In biological study, and in numerous Application Areass, as diagnosis, biotechnology, Forensic Biology, in biosystematics, DNA sequence dna knowledge has become indispensable knowledge.The quick order-checking speed with modern DNA sequencing technology has contributed to reaching the complete DNA sequence dna of order-checking, or polytype gene order-checking and living species, comprise human genome and other many animals, the global DNA sequence of plant and microbial species.
In recent years, sequencing technologies is updated, and novel method is come out one after another, and order-checking scale also constantly expands.The simple operation of sequencing technologies, with low costization and high-throughput change into the developing direction into sequencing technologies.SOLiD, 454 and the high throughput sequencing technologies of new generation that is representative such as Solexa, divide the world of sequencing equally with the attitude of three state's tripartite confrontations.Three kinds of technology are each has something to recommend him, and the speed that development upgrades is with rapid changepl. never-ending changes and improvements.But presently, the process prepared of its sequencing library is all still comparatively complicated.
The preparation of sequencing library as first in whole sequencing procedure and and important step, have conclusive effect to the quality of sequencing result.The preparation of sequencing library generally comprises following step.The first step how to be extracted in high quality from sample to be detected by nucleic acid.For different samples, the experiment flow selected by people and mode will have larger difference; Second step how the nucleic acid of long segment is carried out the process of small segment flower, mainly for the RNA equal samples of genomic dna and long segment, because sequenator is for reading long restriction at present, can only check order to the random small segment of preparation, spliced by bioinformatics method again, draw the sequence information of full-length genome.Current nucleic acid small segmentization uses physical action to carry out substantially, and ultrasonic wave becomes due to the various advantage of himself universal way smashing DNA, by the small pieces segment DNA regulating ultrasonic power and ultrasonic time to obtain different lengths.3rd step how the two ends of small segment nucleic acid is connected the universal linker sequence needed for above checking order; 4th step how the nucleotide sequence being connected with joint sequence is carried out the amplification of unit molecule multiple copied, includes emulsion-based PCR, bridge-type PCR and rolling circle amplification etc., guarantees the raw information of real reflected sample while expanding order-checking strength of signal.And emulsion-based PCR generally adopts microballoon to catch a template DNA to a bead, utilize emulsion-based PCR to increase single template, same template is increased on a microballoon up to a million templates clones.
In general, the microballoon adopted in emulsion-based PCR is polystyrene (PS) microballoon, and carries out Streptavidin modification at microsphere surface, thus makes it can catch biotin labeled DNA molecular.At present, the method for synthesizing Monodisperse Polystyrene Microspheres has microemulsion polymerization method, emulsion polymerization, dispersion copolymerization method, suspension polymerization and seeded polymerization etc.But existing synthetic method be all linking agent and monomer mixing after be polymerized again, the polymer microballoon polymkeric substance of this way synthesis is overall crosslinked, often sphericity is poor, and size tunable is low, the requirement thus usually needing sorting just can reach size distribution deviation to be less than 5%.In order to synthesize the microballoon obtaining being cross-linked, but do not have influence on sphericity and the monodispersity of microballoon, although Japanese Patent JP58-106554 and JP63-191818 proposes the method for seeding polymerization, namely first obtain seed by letex polymerization, then increase, expand particle.The shortcoming of present method be microballoon in process of growth, produce secondary particle, need screening removing, cause product yield to reduce, complicated operation, less economical.Therefore, it is very difficult that the micron size that obtain uniform particle diameter has crosslinked polymer microballoon particle.
Due to after needing surface amination for the PS microballoon of nucleic acid amplification could and Streptavidin be cross-linked, thus to be connected with the DNA profiling being marked with vitamin H, and then to carry out emulsion-based PCR amplification.Therefore, the PS microballoon preparing amino content high is vital, at present, when preparing amination polystyrene microsphere by polymerization, all adopts and adds acrylic acid mode to Surfaces of Polystyrene Microparticles introducing amino.But adopt the amination polystyrene microsphere major part amino prepared in this way and be embedded in microballoon inside, the amino distribution proportion of Surfaces of Polystyrene Microparticles is not high.
Summary of the invention
An object of the present invention is a kind of preparation method of surface amination polystyrene microsphere, comprise the following steps:
(1) dispersion agent and styrene monomer are added in medium, pass into nitrogen, stir;
(2) heat temperature raising, adds initiator, and Keep agitation under constant temperature, obtains polystyrene microsphere;
(3) get another container and add 3-aminopropyl triethoxysilane and solvent, vortex shakes, and adds the polystyrene microsphere of preparation, passes into nitrogen, calorify isothermal reaction, obtain amination polystyrene microsphere;
(4) get amination polystyrene microsphere, add after ethanesulfonic acid buffer mixes and add linking agent, stir, centrifuge washing, removes supernatant, precipitates, vibration resuspended through PBS, namely obtains the polystyrene microsphere that activates after supersound process; Get Streptavidin to be dissolved in PBS damping fluid, join in the polystyrene microsphere solution of overactivation, centrifugal, the microballoon after precipitation PBS repeated washing is resuspended in PBS damping fluid through ultrasonic disperse, adds Methionin, and room temperature is slightly shaken, centrifuge washing;
(5) use first alcohol and water cleaning microballoon respectively, to obtain final product.
Described medium is first alcohol and water.
Described initiator is one or more in Diisopropyl azodicarboxylate, acetyl peroxide and cyclohexanone peroxide, and preferably Diisopropyl azodicarboxylate and cyclohexanone peroxide weight ratio are 4:1.
Described linking agent is tetramethylol methane tetraacrylate and double pentaerythritol methacrylate, volume ratio (5-6): 1, is preferably 6:1.
Described dispersion agent is made up of Polyvinylpyrolidone (PVP), alkylphenol polyoxyethylene-10 and polyoxyethylene glycol.
The weight percent of described styrene monomer, dispersion agent, initiator and medium is followed successively by the styrene monomer of 33-34%, the dispersion agent of 0.2-0.25%, the initiator of 0.035%-0.045, and surplus is medium.Be preferably the styrene monomer of 34%, the dispersion agent of 0.25%, the initiator of 0.04%, surplus is medium.
Described solvent is made up of oil of mirbane and methylene dichloride, and the volume ratio of oil of mirbane and methylene dichloride is 1:7.
The described gauge adding the polystyrene microsphere of preparation is 0.4-0.6g/ml3-aminopropyl triethoxysilane.
The speed of described stirring is 150 ~ 180 revs/min.
Method mild condition provided by the invention, productive rate are high, and preparation method is simple, is suitable for applying on a large scale.Meanwhile, invention increases the amino distribution proportion at Surfaces of Polystyrene Microparticles.The use of linking agent tetramethylol methane tetraacrylate and double pentaerythritol methacrylate, microballoon physical strength prepared by the present invention is comparatively large, is easy to reclaim.In addition, by reasonably optimizing the medium in reaction system, initiator and dispersion agent, the microspherulite diameter obtained is even, can meet the needs of gene sequencing.
Embodiment
embodiment 1
(1) 0.3g Polyvinylpyrolidone (PVP), 1.2g alkylphenol polyoxyethylene-10,1.625g polyoxyethylene glycol and 425g styrene monomer are added in the mixture of 400g water and 421.375g ethylene glycol, pass into nitrogen, stir 25 minutes with 150rpm/min;
(2) be heated to 70 DEG C, add 0.4g Diisopropyl azodicarboxylate and 0.1g cyclohexanone peroxide, with 180rpm/min Keep agitation 10 hours under constant temperature, obtain polystyrene microsphere;
(3) get another container and add 20ml3-aminopropyl triethoxysilane and 35ml solvent (volume ratio of oil of mirbane and methylene dichloride is 1:7), vortex shakes 15 minutes, add the 10g polystyrene microsphere of preparation, pass into nitrogen, calorify 65 DEG C of isothermal reactions 7 hours, obtain amination polystyrene microsphere;
(4) 4.5mg amination polystyrene microsphere is got, add after 0.9ml ethanesulfonic acid buffer mixes and add tetramethylol methane tetraacrylate and double pentaerythritol methacrylate activation, 160rpm/min stirs 15 minutes, 12000rpm/min centrifuge washing, remove supernatant, precipitate, vibration resuspended through PBS, namely obtain the polystyrene microsphere that activates after supersound process; Get Streptavidin to be dissolved in PBS damping fluid, join in the polystyrene microsphere solution of overactivation, 12000rpm/min is centrifugal, microballoon after precipitation PBS repeated washing is resuspended in PBS damping fluid through ultrasonic disperse, add Methionin, room temperature is slightly shaken, centrifuge washing;
(5) use first alcohol and water cleaning microballoon respectively, to obtain final product.
embodiment 2
(1) 0.6g Polyvinylpyrolidone (PVP), 1g alkylphenol polyoxyethylene-10,0.9g polyoxyethylene glycol and 412.5g styrene monomer are added in the mixture of 340g water and 494.56g ethylene glycol, pass into nitrogen, stir 30 minutes with 160rpm/min;
(2) be heated to 70 DEG C, add 0.25g acetyl peroxide and 0.19g cyclohexanone peroxide, with 160rpm/min Keep agitation 9 hours under constant temperature, obtain polystyrene microsphere;
(3) get another container and add 20ml3-aminopropyl triethoxysilane and 35ml solvent (volume ratio of oil of mirbane and methylene dichloride is 1:7), vortex shakes 10 minutes, add the 8g polystyrene microsphere of preparation, pass into nitrogen, calorify 62 DEG C of isothermal reactions 6 hours, obtain amination polystyrene microsphere;
(4) 4.5mg amination polystyrene microsphere is got, add after 0.8ml ethanesulfonic acid buffer mixes and add tetramethylol methane tetraacrylate and double pentaerythritol methacrylate activation, 170rpm/min stirs 15 minutes, 12000rpm/min centrifuge washing, remove supernatant, precipitate, vibration resuspended through PBS, namely obtain the polystyrene microsphere that activates after supersound process; Get Streptavidin to be dissolved in PBS damping fluid, join in the polystyrene microsphere solution of overactivation, 12000rpm/min is centrifugal, microballoon after precipitation PBS repeated washing is resuspended in PBS damping fluid through ultrasonic disperse, add Methionin, room temperature is slightly shaken, centrifuge washing;
(5) use first alcohol and water cleaning microballoon respectively, to obtain final product.
embodiment 3
(1) 0.8g Polyvinylpyrolidone (PVP), 0.75g alkylphenol polyoxyethylene-10,1.2g polyoxyethylene glycol and 417.5g styrene monomer are added in the mixture of 280g water and 549.19g ethylene glycol, pass into nitrogen, stir 30 minutes with 170rpm/min;
(2) be heated to 70 DEG C, add 0.56g acetyl peroxide, with 160rpm/min Keep agitation 9 hours under constant temperature, obtain polystyrene microsphere;
(3) get another container and add 20ml3-aminopropyl triethoxysilane and 33ml solvent (volume ratio of oil of mirbane and methylene dichloride is 1:7), vortex shakes 10 minutes, add the 12g polystyrene microsphere of preparation, pass into nitrogen, calorify 60 DEG C of isothermal reactions 8 hours, obtain amination polystyrene microsphere;
(4) 4.5mg amination polystyrene microsphere is got, add after 0.8ml ethanesulfonic acid buffer mixes and add tetramethylol methane tetraacrylate and double pentaerythritol methacrylate activation, 165rpm/min stirs 15 minutes, 12000rpm/min centrifuge washing, remove supernatant, precipitate, vibration resuspended through PBS, namely obtain the polystyrene microsphere that activates after supersound process; Get Streptavidin to be dissolved in PBS damping fluid, join in the polystyrene microsphere solution of overactivation, 12000rpm/min is centrifugal, microballoon after precipitation PBS repeated washing is resuspended in PBS damping fluid through ultrasonic disperse, add Methionin, room temperature is slightly shaken, centrifuge washing;
(5) use first alcohol and water cleaning microballoon respectively, to obtain final product.
embodiment 4
(1) 0.8g Polyvinylpyrolidone (PVP), 0.8g alkylphenol polyoxyethylene-10,1.2g polyoxyethylene glycol and 417.5g styrene monomer are added in the mixture of 280g water and 549.19g ethylene glycol, pass into nitrogen, stir 30 minutes with 165rpm/min;
(2) be heated to 70 DEG C, add 0.51g cyclohexanone peroxide, with 175rpm/min Keep agitation 9 hours under constant temperature, obtain polystyrene microsphere;
(3) get another container and add 20ml3-aminopropyl triethoxysilane and 32ml solvent (volume ratio of oil of mirbane and methylene dichloride is 1:7), vortex shakes 10 minutes, add the 11g polystyrene microsphere of preparation, pass into nitrogen, calorify 60 DEG C of isothermal reactions 8 hours, obtain amination polystyrene microsphere;
(4) 4.5mg amination polystyrene microsphere is got, add after 0.8ml ethanesulfonic acid buffer mixes and add tetramethylol methane tetraacrylate and double pentaerythritol methacrylate activation, 150rpm/min stirs 15 minutes, 12000rpm/min centrifuge washing, remove supernatant, precipitate, vibration resuspended through PBS, namely obtain the polystyrene microsphere that activates after supersound process; Get Streptavidin to be dissolved in PBS damping fluid, join in the polystyrene microsphere solution of overactivation, 12000rpm/min is centrifugal, microballoon after precipitation PBS repeated washing is resuspended in PBS damping fluid through ultrasonic disperse, add Methionin, room temperature is slightly shaken, centrifuge washing;
(5) use first alcohol and water cleaning microballoon respectively, to obtain final product.
Above-mentioned detailed description is illustrating for one of them possible embodiments of the present invention, and this embodiment is also not used to limit the scope of the claims of the present invention, and the equivalence that all the present invention of disengaging do is implemented or changed, and all should be contained in the scope of technical solution of the present invention.
Claims (9)
1. a preparation method for surface amination polystyrene microsphere, is characterized in that, comprises the following steps:
Dispersion agent and styrene monomer are added in medium, passes into nitrogen, stir;
Heat temperature raising, adds initiator, and Keep agitation under constant temperature, obtains polystyrene microsphere;
Get another container and add 3-aminopropyl triethoxysilane and solvent, vortex shakes, and adds the polystyrene microsphere of preparation, passes into nitrogen, calorify isothermal reaction, obtain amination polystyrene microsphere;
Get amination polystyrene microsphere, add after ethanesulfonic acid buffer mixes and add linking agent, stir, centrifuge washing, removes supernatant, precipitates, vibration resuspended through PBS, namely obtains the polystyrene microsphere that activates after supersound process; Get Streptavidin to be dissolved in PBS damping fluid, join in the polystyrene microsphere solution of overactivation, centrifugal, the microballoon after precipitation PBS repeated washing is resuspended in PBS damping fluid through ultrasonic disperse, adds Methionin, and room temperature is slightly shaken, centrifuge washing;
Use first alcohol and water cleaning microballoon respectively, to obtain final product.
2. the preparation method of surface amination polystyrene microsphere as claimed in claim 1, it is characterized in that, described medium is first alcohol and water.
3. the preparation method of surface amination polystyrene microsphere as claimed in claim 1, it is characterized in that, described initiator is one or more in Diisopropyl azodicarboxylate, acetyl peroxide and cyclohexanone peroxide, and preferably Diisopropyl azodicarboxylate and cyclohexanone peroxide weight ratio are 4:1.
4. the preparation method of surface amination polystyrene microsphere as claimed in claim 1, it is characterized in that, described linking agent is tetramethylol methane tetraacrylate and double pentaerythritol methacrylate, volume ratio (5-6): 1, is preferably 6:1.
5. the preparation method of surface amination polystyrene microsphere as claimed in claim 1, it is characterized in that, described dispersion agent is made up of Polyvinylpyrolidone (PVP), alkylphenol polyoxyethylene-10 and polyoxyethylene glycol.
6. the preparation method of surface amination polystyrene microsphere as claimed in claim 1, it is characterized in that, the weight percent of described styrene monomer, dispersion agent, initiator and medium is followed successively by the styrene monomer of 33-34%, the dispersion agent of 0.2-0.25%, the initiator of 0.035%-0.045, surplus is medium; Be preferably the styrene monomer of 34%, the dispersion agent of 0.25%, the initiator of 0.04%, surplus is medium.
7. the preparation method of surface amination polystyrene microsphere as claimed in claim 1, it is characterized in that, described solvent is made up of oil of mirbane and methylene dichloride, and the volume ratio of oil of mirbane and methylene dichloride is 1:7.
8. the preparation method of surface amination polystyrene microsphere as claimed in claim 1, is characterized in that, described in add the polystyrene microsphere of preparation gauge be 0.4-0.6g/ml3-aminopropyl triethoxysilane.
9. the preparation method of surface amination polystyrene microsphere as claimed in claim 1, it is characterized in that, the speed of described stirring is 150 ~ 180 revs/min.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111088033A (en) * | 2019-12-25 | 2020-05-01 | 苏州为度生物技术有限公司 | Preparation method of monodisperse high-performance quantum dot fluorescent microspheres |
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| CN102649826A (en) * | 2011-02-25 | 2012-08-29 | 常州均益新材料科技有限公司 | Preparation method of particle diameter controllable monodisperse polystyrene microspheres |
| CN103439515A (en) * | 2013-08-14 | 2013-12-11 | 南方医科大学 | Method for detecting valence of antibody |
| CN104804114A (en) * | 2015-04-28 | 2015-07-29 | 北京中科紫鑫科技有限责任公司 | Preparation method of streptavidin-modified polystyrene microspheres for nucleic acid amplification |
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2099000A (en) * | 1981-04-06 | 1982-12-01 | Badische Yuka Co Ltd | Inorganic filler-containing vinyl monomer compositions and process for the production therefrom of polymer particles |
| CN101672847A (en) * | 2009-09-30 | 2010-03-17 | 青岛科技大学 | Preparation method of protein chip glass carrier |
| CN102649826A (en) * | 2011-02-25 | 2012-08-29 | 常州均益新材料科技有限公司 | Preparation method of particle diameter controllable monodisperse polystyrene microspheres |
| CN102358783A (en) * | 2011-07-27 | 2012-02-22 | 武汉大学 | Preparation method of polystyrene/gold composite microspheres |
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| CN111088033A (en) * | 2019-12-25 | 2020-05-01 | 苏州为度生物技术有限公司 | Preparation method of monodisperse high-performance quantum dot fluorescent microspheres |
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Application publication date: 20151209 |