CN104804114A - Preparation method of streptavidin-modified polystyrene microspheres for nucleic acid amplification - Google Patents
Preparation method of streptavidin-modified polystyrene microspheres for nucleic acid amplification Download PDFInfo
- Publication number
- CN104804114A CN104804114A CN201510208171.8A CN201510208171A CN104804114A CN 104804114 A CN104804114 A CN 104804114A CN 201510208171 A CN201510208171 A CN 201510208171A CN 104804114 A CN104804114 A CN 104804114A
- Authority
- CN
- China
- Prior art keywords
- preparation
- polystyrene microsphere
- streptavidin
- reaction
- initiator
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Compositions Of Macromolecular Compounds (AREA)
- Processes Of Treating Macromolecular Substances (AREA)
- Manufacturing Of Micro-Capsules (AREA)
Abstract
The invention belongs to the field of genome sequencing, and particularly relates to a preparation method of streptavidin-modified polystyrene microspheres for nucleic acid amplification. The preparation method comprises the following steps: firstly, preparing polystyrene microspheres of which the grain diameter is 25 microns by a dispersion polymerization method; performing carboxylation on the surfaces of polystyrene microspheres; performing crosslinking on the carboxylated polystyrene microspheres and streptavidin so as to obtain the streptavidin-modified polystyrene microspheres. According to the preparation method disclosed by the invention, monodisperse PS microspheres are successfully prepared by adopting the dispersion polymerization method, the grain diameter is 25 microns, and the dispersion coefficient is low; the surface carboxylation of the PS microspheres and the modifying reaction conditions of the streptavidin are systematically optimized, so that polystyrene microspheres with higher intake of streptavidin are obtained.
Description
Technical field
The invention belongs to gene order-checking field, be specifically related to a kind of preparation method being applied to the carboxylic polystyrene microsphere of gene order-checking nucleic acid amplification.
Background technology
As one of most important invention of life science in last century, DNA sequencing technology profoundly changes people to the understanding of life quintessence and control ability, has greatly promoted the development of global life science research.Were it not for sequencing technologies, gene order just cannot be determined, the investigative techniques such as enzyme is cut, clone, reverse transcription, cDNA, PCR, SNP, RNAi are not just known where to begin at all yet, and life science does not have the flourish of today.The life science GenBank that All the world knows, and the Human Genome Project that the global cooperation lasting 13 years costs 300,000,000 dollars completes, be based upon on the basis of order-checking invariably.
Since Sanger in 1977 has invented the technology of the two deoxidations termination principles mensuration nucleotide sequences that make use of archaeal dna polymerase, sequencing technologies has been 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 carboxyl groups 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 carboxyl-content high is vital, at present, when preparing carboxylic polystyrene microsphere by polymerization, all adopts the acrylic acid mode of interpolation to introduce carboxyl to Surfaces of Polystyrene Microparticles.But adopt the carboxylic polystyrene microsphere major part carboxyl prepared in this way and be embedded in microballoon inside, the carboxyl distribution proportion of Surfaces of Polystyrene Microparticles is not high, is generally 0.25mmol/g.
Summary of the invention
An object of the present invention is to provide the preparation method of the polystyrene microsphere that a kind of Streptavidin is modified, its microballoon prepared can be used for the nucleic acid amplification in the preparation of gene order-checking library, and described microspherulite diameter is 25 microns, and concrete steps are as follows:
(1) dispersion agent is added in reaction medium, pass into nitrogen; Be stirred to homogeneous system;
(2) styrene monomer and initiator are added in reaction system; Reaction system is heated up; Then isothermal reaction is carried out under agitation; Be cooled to room temperature, washing, drying, obtain polystyrene microsphere;
(3) get another container disease and add Tetra hydro Phthalic anhydride (PA) powder and a certain amount of solvent, sonic oscillation 10min, then polystyrene microsphere is added, in reaction flask after logical purity nitrogen for some time, aluminum chloride is added under whipped state, react 6 hours at 60 DEG C after logical purity nitrogen 3min again, obtain carboxylic polystyrene microsphere.
(4) carboxylic polystyrene microsphere is got, add after ethanesulfonic acid buffer (MES) mixes and add water-soluble carbodiimide class linking agent (EDC) and N-hydroxy-succinamide (NHS) activation, EDC concentration 1.0-1.5g/L, between EDC and NHS ratio 1:1, mild stirring under room temperature condition, time controling is at 20min; Centrifuge washing, removes supernatant, precipitates, vibration resuspended through 0.1mol/LPBS, namely obtains the polystyrene microsphere that activates after supersound process; Get Streptavidin to be dissolved in the PBS damping fluid of pH6.7, join in the polystyrene microsphere solution of overactivation, mild stirring under room temperature condition, reaction times 3h; The centrifugal 20min of 13000g, precipitation is resuspended in PBS damping fluid through ultrasonic disperse with containing the microballoon after the PBS repeated washing of 0.05%Tween20, and add Methionin, room temperature slightly shakes 30min, and centrifuge washing to obtain final product.
In one embodiment of the invention, described reaction medium is selected from the one or more kinds of arbitrary combination of water, ethanol, ethylene glycol monomethyl ether and methyl alcohol.In preferred embodiments, described reaction medium is water and ethylene glycol monomethyl ether, and wherein, the weight percent of water and ethylene glycol monomethyl ether is 10 ~ 90% and 10 ~ 90%; Also can be 20 ~ 40% and 60 ~ 80%, be preferably 23% and 77%.
Described dispersion agent is the one or more kinds of arbitrary combination in sodium phosphate, polyvinylpyrrolidone, polyoxyethylene glycol, polyacrylic acid, polyvinyl alcohol and hydroxypropylcellulose.In preferred embodiments, described dispersion agent is made up of polyvinyl alcohol, hydroxypropylcellulose and polyoxyethylene glycol, and the weight percent of polyvinyl alcohol, hydroxypropylcellulose and polyoxyethylene glycol is 10 ~ 80%, 10 ~ 80% and 10 ~ 80%; Also can be 20 ~ 40%, 30 ~ 50% and 20 ~ 50%, be preferably 25%, 35% and 40%.
Initiator is selected from one or both the arbitrary proportion combination of Diisopropyl azodicarboxylate (AIBN) or benzoyl peroxide (BPO).In preferred embodiments, described initiator is Diisopropyl azodicarboxylate.
In another embodiment of the invention, in final reaction system, the weight percent of styrene monomer, dispersion agent, initiator and reaction medium is followed successively by the styrene monomer of 5 ~ 40%, the dispersion agent of 0.1 ~ 1%, the initiator of 0.025 ~ 0.125%, reaction medium surplus; Be preferably the styrene monomer of 32%, the dispersion agent of 0.3%, the initiator of 0.05%, reaction medium surplus.
In the present invention further embodiment, reaction system is warming up to 10-90 DEG C by described step (2), is preferably 75 DEG C; Stirring velocity controls at 80 ~ 120 revs/min, is preferably 100 revs/min.
In another embodiment of the invention, in step (3), 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 weight ratio of polystyrene microsphere, Tetra hydro Phthalic anhydride and aluminum chloride is 1:0.02:0.05.
In a preferred embodiment of the present invention, in step (4), the condition of described centrifuge washing is the centrifugal 20min of rotating speed 13000g, precipitates three times by 0.1mol/L phosphoric acid buffer (PBS) repeated washing; The weight ratio of described carboxylic polystyrene microsphere and Streptavidin is 1:0.5.
In gene order-checking, the particle diameter for the PS microballoon of DNA library amplifying nucleic acid amplification is generally 25 microns, and requires that monodispersity is good.But in PS method for preparing microsphere, PS microballoon monodispersity prepared by dispersion copolymerization method is good, and the particle diameter of microballoon is less, although the PS microspherulite diameter of seeded polymerization and suspension polymerization preparation is comparatively large, but the monodispersity of microballoon is poor, and dispersion coefficient is high.Therefore in the art, preparing particle diameter is that the Monodisperse Polystyrene Microspheres of 20 to 50 microns also exists the problems referred to above always.The present invention is groped by lot of experiments and unexpected discovery, and under the specific reaction conditions of one, adopt dispersion copolymerization method successfully to prepare Monodisperse Polystyrene Microspheres, particle diameter is 25 microns and dispersion coefficient is low.In addition, owing to needing to carry out Streptavidin modification when PS microballoon is used for emulsion-based PCR amplification, and PS microballoon to need to carry out after surface carboxyl groups could and Streptavidin crosslinked, therefore, the present invention has carried out systemic optimization to the reaction conditions that the surface carboxyl groups of PS microballoon and Streptavidin are modified, thus obtains the higher polystyrene microsphere of a kind of Streptavidin introduction volume.
Embodiment
Further will illustrate the present invention below.It is pointed out that following explanation is only illustrating the technical scheme that application claims is protected, any restriction not to these technical schemes.The content that protection scope of the present invention is recorded with appended claims is as the criterion.
The preparation of embodiment 1 polystyrene microsphere
The polyoxyethylene glycol of the water of 230g, the ethylene glycol monomethyl ether of 770g, the polyvinyl alcohol of 1.1g, the hydroxypropylcellulose of 1.54g and 1.76g is added in reaction vessel; pass into nitrogen protection; and at room temperature stir 20 minutes to homogeneous system, then add styrene monomer 473g and Diisopropyl azodicarboxylate 0.73g.Be warming up to 75 DEG C, isothermal reaction 8 hours.Reaction terminates, and be cooled to room temperature, product is centrifugal, and uses ethanol repetitive scrubbing, dry, obtains Monodisperse Polystyrene Microspheres.
The preparation of embodiment 2 polystyrene microsphere
The polyoxyethylene glycol of the water of 230g, the ethylene glycol monomethyl ether of 770g, the polyvinyl alcohol of 1.0g, the hydroxypropylcellulose of 1.59g and 1.81g is added in reaction vessel; pass into nitrogen protection; and at room temperature stir 20 minutes to homogeneous system, then add styrene monomer 473g and Diisopropyl azodicarboxylate 0.81g.Be warming up to 75 DEG C, isothermal reaction 12 hours.Reaction terminates, and be cooled to room temperature, product is centrifugal, and uses ethanol repetitive scrubbing, dry, obtains Monodisperse Polystyrene Microspheres.
The preparation of embodiment 3 polystyrene microsphere
The polyoxyethylene glycol of the water of 230g, the ethylene glycol monomethyl ether of 770g, the hydroxypropylcellulose of 2.64g and 1.76g is added in reaction vessel; pass into nitrogen protection; and at room temperature stir 20 minutes to homogeneous system, then add styrene monomer 473g and Diisopropyl azodicarboxylate 0.73g.Be warming up to 75 DEG C, isothermal reaction 8 hours.Reaction terminates, and be cooled to room temperature, product is centrifugal, and uses ethanol repetitive scrubbing, dry, obtains Monodisperse Polystyrene Microspheres.
The preparation of embodiment 4 polystyrene microsphere
The polyoxyethylene glycol of the water of 230g, the ethylene glycol monomethyl ether of 770g, the polyvinyl alcohol of 1.2g, the hydroxypropylcellulose of 1.74g and 1.86g is added in reaction vessel; pass into nitrogen protection; and at room temperature stir 20 minutes to homogeneous system, then add styrene monomer 473g and Diisopropyl azodicarboxylate 0.73g.Be warming up to 75 DEG C, isothermal reaction 8 hours.Reaction terminates, and be cooled to room temperature, product is centrifugal, and uses ethanol repetitive scrubbing, dry, obtains Monodisperse Polystyrene Microspheres.
The preparation of embodiment 5 polystyrene microsphere
The polyoxyethylene glycol of the water of 230g, the ethylene glycol monomethyl ether of 770g, the polyvinyl alcohol of 1.2g, the hydroxypropylcellulose of 1.74g and 1.86g is added in reaction vessel; pass into nitrogen protection; and at room temperature stir 20 minutes to homogeneous system, then add styrene monomer 473g and Diisopropyl azodicarboxylate 0.6g.Be warming up to 75 DEG C, isothermal reaction 8 hours.Reaction terminates, and be cooled to room temperature, product is centrifugal, and uses ethanol repetitive scrubbing, dry, obtains Monodisperse Polystyrene Microspheres.
The preparation of embodiment 6 polystyrene microsphere
The polyoxyethylene glycol of the water of 230g, the ethylene glycol monomethyl ether of 770g, the polyvinyl alcohol of 1.2g, the hydroxypropylcellulose of 1.74g and 1.86g is added in reaction vessel; pass into nitrogen protection; and at room temperature stir 20 minutes to homogeneous system, then add styrene monomer 473g and Diisopropyl azodicarboxylate 0.85g.Be warming up to 75 DEG C, isothermal reaction 8 hours.Reaction terminates, and be cooled to room temperature, product is centrifugal, and uses ethanol repetitive scrubbing, dry, obtains Monodisperse Polystyrene Microspheres.
The preparation of embodiment 7 polystyrene microsphere
The polyoxyethylene glycol of the water of 200g, the ethylene glycol monomethyl ether of 800g, the polyvinyl alcohol of 1.1g, the hydroxypropylcellulose of 1.54g and 1.76g is added in reaction vessel; pass into nitrogen protection; and at room temperature stir 20 minutes to homogeneous system, then add styrene monomer 473g and Diisopropyl azodicarboxylate 0.73g.Be warming up to 75 DEG C, isothermal reaction 8 hours.Reaction terminates, and be cooled to room temperature, product is centrifugal, and uses ethanol repetitive scrubbing, dry, obtains Monodisperse Polystyrene Microspheres.
The preparation of embodiment 8 polystyrene microsphere
The polyoxyethylene glycol of the water of 260g, the ethylene glycol monomethyl ether of 740g, the polyvinyl alcohol of 1.1g, the hydroxypropylcellulose of 1.54g and 1.76g is added in reaction vessel; pass into nitrogen protection; and at room temperature stir 20 minutes to homogeneous system, then add styrene monomer 473g and Diisopropyl azodicarboxylate 0.73g.Be warming up to 75 DEG C, isothermal reaction 8 hours.Reaction terminates, and be cooled to room temperature, product is centrifugal, and uses ethanol repetitive scrubbing, dry, obtains Monodisperse Polystyrene Microspheres.
The preparation of embodiment 9 polystyrene microsphere
The polyoxyethylene glycol of the water of 230g, the ethylene glycol monomethyl ether of 770g, the polyvinyl alcohol of 1.1g, the hydroxypropylcellulose of 1.54g and 1.76g is added in reaction vessel; pass into nitrogen protection; and at room temperature stir 20 minutes to homogeneous system, then add styrene monomer 450g and Diisopropyl azodicarboxylate 0.73g.Be warming up to 75 DEG C, isothermal reaction 8 hours.Reaction terminates, and be cooled to room temperature, product is centrifugal, and uses ethanol repetitive scrubbing, dry, obtains Monodisperse Polystyrene Microspheres.
The preparation of embodiment 10 polystyrene microsphere
The polyoxyethylene glycol of the water of 230g, the ethylene glycol monomethyl ether of 770g, the polyvinyl alcohol of 1.1g, the hydroxypropylcellulose of 1.54g and 1.76g is added in reaction vessel; pass into nitrogen protection; and at room temperature stir 20 minutes to homogeneous system, then add styrene monomer 500g and Diisopropyl azodicarboxylate 0.73g.Be warming up to 75 DEG C, isothermal reaction 8 hours.Reaction terminates, and be cooled to room temperature, product is centrifugal, and uses ethanol repetitive scrubbing, and dry, obtain Monodisperse Polystyrene Microspheres, particle diameter is 25.0 μm, dispersion coefficient 1.7%.
The sign of polystyrene microsphere prepared by embodiment 1-10
JSM-6700F scanning electronic microscope is adopted to measure the particle diameter of obtained PS microballoon and size distribution.With 100 microballoons for benchmark, the median size of measuring and calculating microballoon and size distribution, formula is as follows:
f
s＝δ/d (3)
In formula: δ is standard variance; d
ifor the diameter of single particle; D is the mean diameter of particle; N is number of particles, f
sfor dispersion coefficient, concrete outcome is as follows:
| Particle diameter | Dispersion coefficient | |
| Embodiment 1 | 25μm | 0.019 |
| Embodiment 2 | 25μm | 0.028 |
| Embodiment 3 | 18μm | 0.051 |
| Embodiment 4 | 24μm | 0.064 |
| Embodiment 5 | 25μm | 0.057 |
| Embodiment 6 | 25μm | 0.092 |
| Embodiment 7 | 28μm | 0.049 |
| Embodiment 8 | 22μm | 0.063 |
| Embodiment 9 | 23μm | 0.071 |
| Embodiment 10 | 29μm | 0.068 |
The preparation of embodiment 11 carboxylic polystyrene microsphere
Tetra hydro Phthalic anhydride powder and the 35ml solvent (volume ratio of oil of mirbane and methylene dichloride is 1:7) of 0.2g is added in three-necked bottle, sonic oscillation 10min, then the polystyrene microsphere of 10g is added, in reaction flask after logical purity nitrogen for some time, under whipped state, add 0.5g aluminum chloride, then react 6 hours at 60 DEG C after logical purity nitrogen 3min.
The titration of embodiment 12 carboxylic polystyrene microsphere
Get a certain amount of carboxylated PS microballoon and PS microballoon (blank), in two kinds of microballoons, move into NaOH and dehydrated alcohol, preserve under room temperature and spend the night, be then titrated to neutrality with HCl; Calculate the carboxyl loading (mmol/g) of carboxylated PS microballoon.
In burette test, another interpolation 4 comparative examples, comparative example is except following table parameter, and other is all with embodiment 11, and parameter sees the following form:
The titration results of carboxylic polystyrene microsphere prepared by embodiment 11 and comparative example 1-4 is as follows:
| Carboxyl loading (mmol/g) | |
| Comparative example 1 | 0.7 |
| Comparative example 2 | 0.3 |
| Comparative example 3 | 0.9 |
| Comparative example 4 | 1.1 |
| Embodiment 11 | 2.2 |
Embodiment 13 Streptavidin modifies the preparation of polystyrene microsphere
1) the carboxylated PS microballoon of 4mg is got, add after 0.9mL ethanesulfonic acid buffer (MES) mixes and add water-soluble carbodiimide class linking agent (EDC) and N-hydroxy-succinamide (NHS) activation, EDC concentration 1.2g/L, between EDC and NHS ratio 1:1, mild stirring under room temperature condition, time controling is at 20min;
2) centrifuge washing, the centrifugal 20min of rotating speed 13000g, precipitates three times by 0.1mol/L phosphoric acid buffer (PBS) repeated washing, removes supernatant, precipitates, vibration resuspended through 0.1mol/L PBS, namely obtains the latex microsphere that activates after supersound process;
3) get 2mg Streptavidin to be dissolved in the PBS damping fluid of pH6.7, join 1mL in the latex microsphere solution of overactivation, mild stirring under room temperature condition, reaction times 3h;
4) after reaching the reaction times, the centrifugal 20min of 13000g, precipitation is with being resuspended in PBS damping fluid containing the microballoon after the PBS repeated washing of 0.05%Tween20 through ultrasonic disperse, add 30nmol Methionin, room temperature slightly shakes 30min, the centrifugal 20min of 13000g washs three times, ultrasonic be resuspended in 0.1M pH7.4PBS damping fluid for subsequent use.
Embodiment 14 Streptavidin modifies the Streptavidin assay of polystyrene microsphere
The Streptavidin prepared embodiment 13 by commercially available ELISA kit modifies the Streptavidin content on PS microballoon; In testing process, the consumption of PS microballoon is 1mg, thus measures the Streptavidin content of 1mg PS microballoon, and result shows: embodiment 13 prepares the Streptavidin content 75mg/g that Streptavidin modifies PS microballoon.
Content of the present invention merely illustrates some claimed specific embodiments; one of them or more described technical characteristic can be combined with arbitrary one or more technical scheme in technical scheme; these technical schemes obtained through combination also in the application's protection domain, just as these technical schemes obtained through combination in the disclosure of invention concrete record.
Claims (8)
1. Streptavidin modifies a preparation method for polystyrene microsphere, and its microballoon prepared can be used for the nucleic acid amplification in the preparation of gene order-checking library, and described microspherulite diameter is 25 microns, and concrete steps are as follows:
(1) dispersion agent is added in reaction medium, pass into nitrogen; Be stirred to homogeneous system;
(2) styrene monomer and initiator are added in reaction system; Reaction system is heated up; Then isothermal reaction is carried out under agitation; Be cooled to room temperature, washing, drying.
Described reaction medium is selected from the one or more kinds of arbitrary combination of water, ethanol, ethylene glycol monomethyl ether and methyl alcohol.
Described dispersion agent is the one or more kinds of arbitrary combination in sodium phosphate, polyvinylpyrrolidone, polyoxyethylene glycol, polyacrylic acid, polyvinyl alcohol and hydroxypropylcellulose.
Initiator is selected from one or both the arbitrary proportion combination of Diisopropyl azodicarboxylate (AIBN) or benzoyl peroxide (BPO).
(3) get another container disease and add Tetra hydro Phthalic anhydride (PA) powder and a certain amount of solvent, sonic oscillation 10min, then the polystyrene microsphere of preparation is added, in reaction flask after logical purity nitrogen for some time, aluminum chloride is added under whipped state, react 6 hours at 60 DEG C after logical purity nitrogen 3min again, obtain carboxylic polystyrene microsphere.。
(4) carboxylic polystyrene microsphere is got, add after ethanesulfonic acid buffer (MES) mixes and add water-soluble carbodiimide class linking agent (EDC) and N-hydroxy-succinamide (NHS) activation, EDC concentration 1.0-1.5g/L, between EDC and NHS ratio 1:1, mild stirring under room temperature condition, time controling is at 20min; Centrifuge washing, removes supernatant, precipitates, vibration resuspended through 0.1mol/LPBS, namely obtains the polystyrene microsphere that activates after supersound process; Get Streptavidin to be dissolved in the PBS damping fluid of pH6.7, join in the polystyrene microsphere solution of overactivation, mild stirring under room temperature condition, reaction times 3h; The centrifugal 20min of 13000g, precipitation is resuspended in PBS damping fluid through ultrasonic disperse with containing the microballoon after the PBS repeated washing of 0.05%Tween20, and add Methionin, room temperature slightly shakes 30min, and centrifuge washing to obtain final product.
2. Streptavidin modifies the preparation method of polystyrene microsphere according to claim 1, and it is characterized in that, described reaction medium is water and ethylene glycol monomethyl ether, and wherein, the weight percent of water and ethylene glycol monomethyl ether is 10 ~ 90% and 10 ~ 90%; Also can be 20 ~ 40% and 60 ~ 80%, be preferably 23% and 77%.
3. Streptavidin modifies the preparation method of polystyrene microsphere according to claim 1, it is characterized in that, described dispersion agent is made up of polyvinyl alcohol, hydroxypropylcellulose and polyoxyethylene glycol, and the weight percent of polyvinyl alcohol, hydroxypropylcellulose and polyoxyethylene glycol is 10 ~ 80%, 10 ~ 80% and 10 ~ 80%; Also can be 20 ~ 40%, 30 ~ 50% and 20 ~ 50%, be preferably 25%, 35% and 40%.
4. Streptavidin modifies the preparation method of polystyrene microsphere according to claim 1, it is characterized in that, initiator is selected from one or both the arbitrary proportion combination of Diisopropyl azodicarboxylate (AIBN) or benzoyl peroxide (BPO).In preferred embodiments, described initiator is Diisopropyl azodicarboxylate.
5. Streptavidin modifies the preparation method of polystyrene microsphere according to claim 1, it is characterized in that, in final reaction system, the weight percent of styrene monomer, dispersion agent, initiator and reaction medium is followed successively by the styrene monomer of 5 ~ 40%, the dispersion agent of 0.1 ~ 1%, the initiator of 0.025 ~ 0.125%, reaction medium surplus; Be preferably the styrene monomer of 32%, the dispersion agent of 0.3%, the initiator of 0.05%, reaction medium surplus.
6. Streptavidin modifies the preparation method of polystyrene microsphere according to claim 1, it is characterized in that, in final reaction system, described step (2) reaction system is warming up to 10-90 DEG C, be preferably 75 DEG C; Stirring velocity controls at 80 ~ 120 revs/min, is preferably 100 revs/min.
7. Streptavidin modifies the preparation method of polystyrene microsphere according to claim 1, and it is characterized in that, in step (3), 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 weight ratio of polystyrene microsphere, Tetra hydro Phthalic anhydride and aluminum chloride is 1:0.02:0.05.
8. Streptavidin modifies the preparation method of polystyrene microsphere according to claim 1, it is characterized in that, in step (4), the condition of described centrifuge washing is the centrifugal 20min of rotating speed 13000g, precipitates three times by 0.1mol/L phosphoric acid buffer (PBS) repeated washing; The weight ratio of described carboxylic polystyrene microsphere and Streptavidin is 1:0.5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510208171.8A CN104804114B (en) | 2015-04-28 | 2015-04-28 | Modify the preparation method of polystyrene microsphere for the Streptavidin of nucleic acid amplification |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510208171.8A CN104804114B (en) | 2015-04-28 | 2015-04-28 | Modify the preparation method of polystyrene microsphere for the Streptavidin of nucleic acid amplification |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104804114A true CN104804114A (en) | 2015-07-29 |
| CN104804114B CN104804114B (en) | 2016-05-04 |
Family
ID=53689333
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510208171.8A Active CN104804114B (en) | 2015-04-28 | 2015-04-28 | Modify the preparation method of polystyrene microsphere for the Streptavidin of nucleic acid amplification |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104804114B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105131156A (en) * | 2015-08-24 | 2015-12-09 | 北京中科紫鑫科技有限责任公司 | Preparation method of surface aminated polystyrene microspheres |
| CN106397818A (en) * | 2016-08-31 | 2017-02-15 | 杨明志 | Preparation method of pre-activated polystyrene microspheres |
| CN110511314A (en) * | 2019-08-30 | 2019-11-29 | 华南理工大学 | A kind of metal-chelating polystyrene nanospheres and the preparation method and application thereof |
| CN111013508A (en) * | 2020-01-02 | 2020-04-17 | 金丽素 | Preparation method of carboxyl biological magnetic beads |
| CN112830495A (en) * | 2020-12-31 | 2021-05-25 | 苏州国纳思新材料科技有限公司 | Preparation method of monodisperse colloid emulsion |
| CN116586048A (en) * | 2023-07-17 | 2023-08-15 | 江苏恰瑞生物科技有限公司 | Modified polystyrene adsorption resin for blood perfusion device and preparation method thereof |
| CN117660441A (en) * | 2023-12-08 | 2024-03-08 | 苏州大学 | Method for modifying biotin at two ends of nucleic acid molecule and method for measuring nucleic acid molecule by single molecule force spectrum |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102375057A (en) * | 2010-08-10 | 2012-03-14 | 复旦大学 | Method for simultaneously detecting a plurality of serum markers by homogeneous fluorescence |
| CN102731698A (en) * | 2012-07-10 | 2012-10-17 | 武汉工程大学 | Method for preparing monodisperse polymer microspheres |
-
2015
- 2015-04-28 CN CN201510208171.8A patent/CN104804114B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102375057A (en) * | 2010-08-10 | 2012-03-14 | 复旦大学 | Method for simultaneously detecting a plurality of serum markers by homogeneous fluorescence |
| CN102731698A (en) * | 2012-07-10 | 2012-10-17 | 武汉工程大学 | Method for preparing monodisperse polymer microspheres |
Non-Patent Citations (1)
| Title |
|---|
| 魏荣卿等: ""硝基苯对傅克酰基化反应制备羧基化聚苯乙烯的影响"", 《化工学报》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105131156A (en) * | 2015-08-24 | 2015-12-09 | 北京中科紫鑫科技有限责任公司 | Preparation method of surface aminated polystyrene microspheres |
| CN106397818A (en) * | 2016-08-31 | 2017-02-15 | 杨明志 | Preparation method of pre-activated polystyrene microspheres |
| CN110511314A (en) * | 2019-08-30 | 2019-11-29 | 华南理工大学 | A kind of metal-chelating polystyrene nanospheres and the preparation method and application thereof |
| CN110511314B (en) * | 2019-08-30 | 2021-09-21 | 华南理工大学 | Metal chelating polystyrene nanosphere and preparation method and application thereof |
| CN111013508A (en) * | 2020-01-02 | 2020-04-17 | 金丽素 | Preparation method of carboxyl biological magnetic beads |
| CN112830495A (en) * | 2020-12-31 | 2021-05-25 | 苏州国纳思新材料科技有限公司 | Preparation method of monodisperse colloid emulsion |
| CN112830495B (en) * | 2020-12-31 | 2023-12-29 | 苏州国纳思新材料科技有限公司 | Preparation method of monodisperse colloidal emulsion |
| CN116586048A (en) * | 2023-07-17 | 2023-08-15 | 江苏恰瑞生物科技有限公司 | Modified polystyrene adsorption resin for blood perfusion device and preparation method thereof |
| CN116586048B (en) * | 2023-07-17 | 2023-09-08 | 江苏恰瑞生物科技有限公司 | Modified polystyrene adsorption resin for blood perfusion device and preparation method thereof |
| CN117660441A (en) * | 2023-12-08 | 2024-03-08 | 苏州大学 | Method for modifying biotin at two ends of nucleic acid molecule and method for measuring nucleic acid molecule by single molecule force spectrum |
| CN117660441B (en) * | 2023-12-08 | 2024-05-28 | 苏州大学 | Method for modifying biotin at two ends of nucleic acid molecule and method for measuring nucleic acid molecule by single molecule force spectrum |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104804114B (en) | 2016-05-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104804114A (en) | Preparation method of streptavidin-modified polystyrene microspheres for nucleic acid amplification | |
| CN104844743B (en) | Preparation method for the carboxylic polystyrene microsphere of nucleic acid amplification | |
| US11193122B2 (en) | Methods and systems for droplet-based single cell barcoding | |
| WO2020198071A1 (en) | Three-dimensional spatial analysis | |
| WO2020190509A9 (en) | Methods for using spatial arrays for single cell sequencing | |
| EP2424901B1 (en) | Monodisperse submicron polymer particles | |
| WO2020123318A9 (en) | Resolving spatial arrays using deconvolution | |
| RU98103394A (en) | MAGNETIC POLYMER PARTICLES BASED ON POLYVINYL ALCOHOL, METHODS FOR PRODUCING AND APPLICATION | |
| JPH0723893B2 (en) | Antibody immobilization method | |
| CN111944191B (en) | Quantum dot fluorescent microsphere and preparation method thereof | |
| Besteti et al. | Core/S hell polymer particles by semibatch combined suspension/E mulsion polymerizations for enzyme immobilization | |
| Cummings et al. | On the use of starch in emulsion polymerizations | |
| Ashley et al. | Dispersive solid-phase imprinting of proteins for the production of plastic antibodies | |
| CN104844742B (en) | Preparation method for the polystyrene microsphere of gene order-checking nucleic acid amplification | |
| JPS604501A (en) | Preparation of polymer latex | |
| CN104892827B (en) | A kind of method preparing magnetic composite microsphere | |
| Xu et al. | Controlling biotinylation of microgels and modeling streptavidin uptake | |
| CN105131156A (en) | Preparation method of surface aminated polystyrene microspheres | |
| Ishihara et al. | Molecular integration on phospholipid polymer-coated magnetic beads for gene expression analysis in cells | |
| CN108384774A (en) | A kind of preparation method of magnetic high-molecular particle | |
| JP2002223793A (en) | Method for controlling catalytic activity | |
| CN105085752B (en) | A kind of method for preparing polystyrene microsphere | |
| US20050054815A1 (en) | Stabilized polymer beads and method of preparation | |
| CN103319732B (en) | Preparation method and application of hydrogel based on rhabdovirus | |
| CN104830827B (en) | A kind of preparation method of magnetic immobilized luciferase |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |