CN113058511B - Preparation method of oligonucleotide carrier microsphere - Google Patents

Preparation method of oligonucleotide carrier microsphere Download PDF

Info

Publication number
CN113058511B
CN113058511B CN202110445594.7A CN202110445594A CN113058511B CN 113058511 B CN113058511 B CN 113058511B CN 202110445594 A CN202110445594 A CN 202110445594A CN 113058511 B CN113058511 B CN 113058511B
Authority
CN
China
Prior art keywords
oligo
polyethylene glycol
pegda
microspheres
oligonucleotide
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.)
Active
Application number
CN202110445594.7A
Other languages
Chinese (zh)
Other versions
CN113058511A (en
Inventor
余子夷
文慧琳
张静
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanjing Tech University
Original Assignee
Nanjing Tech University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Nanjing Tech University filed Critical Nanjing Tech University
Priority to CN202110445594.7A priority Critical patent/CN113058511B/en
Publication of CN113058511A publication Critical patent/CN113058511A/en
Application granted granted Critical
Publication of CN113058511B publication Critical patent/CN113058511B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • B01J13/06—Making microcapsules or microballoons by phase separation
    • B01J13/14—Polymerisation; cross-linking
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Dispersion Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Manufacturing Of Micro-Capsules (AREA)

Abstract

The invention discloses a preparation method of oligonucleotide (Oligo) carrier microspheres, which is characterized in that Oligo @ HB-PEGDA and molecules capable of reacting with HB-PEGDA (hyperbranched polyethylene glycol diacrylate) are mixed to prepare water-in-oil droplets, and the gel microspheres are formed by polymerization and crosslinking by taking the water-in-oil droplets as templates. The Oligo carrier microspheres prepared by the method have good monodispersity and controllable Oligo load amount, and can be widely applied to the fields of single cell analysis, in-vitro diagnosis and the like.

Description

Preparation method of oligonucleotide carrier microsphere
Technical Field
The invention relates to biochemical analysis materials, in particular to a preparation method of oligonucleotide (Oligo) carrier microspheres.
Background
The bioactive supported Microsphere (Microsphere) is spherical particle with particle diameter of 50 nm-2 mm and has reactive group such as-NH2-COOH, -SH, etc. The microspheres have small size, so that the microspheres have obvious surface effects such as good affinity and biocompatibility of materials, easy absorption and migration in organisms and the like, and are widely applied to the fields of cytology, immunology, microbiology, molecular biology, clinical diagnosis and treatment, high-throughput gene detection and the like.
Nucleic acid hybridization using nucleic acids immobilized on a solid phase carrier as probes is of great importance in nucleic acid technology. The principle is that the immobilized single-stranded nucleic acid forms a double-helix structure when cooled in solution with a polynucleotide fragment having a complementary base sequence, thereby achieving the purpose of detection and sequence analysis of specific DNA. Yao Nuo and so on directionally link the 5 'end of oligonucleotide or directly link the 5' end of single-stranded DNA to the surface with methylamino (-CH)2NH2) And preparing a long-chain DNA probe on the PS microspheres with the functional groups. Such probes still have a relatively high coupling efficiency at higher temperatures. Therefore, the functionalized polymer microsphere is an ideal nucleic acid hybridization fixed carrier.
When the microspheres are used in a nucleic acid sequencing method, a nucleic acid sample with sequencing is modified by various biochemical methods and then is quickly and efficiently combined with corresponding reactive groups of the microspheres to realize loading; nucleic acid-loaded microspheres are introduced into the channels of the flow cell and into the corresponding reaction micro-tunnels, and incubated for a fixed time to generate all downstream chemical processing steps that are consistently capable of supporting amplification and sequencing. Compared with other types of nucleic acid loading methods, such as chip coating loading coating, the nucleic acid loading microspheres have the advantages of better controllability, large nucleic acid loading capacity and the like.
Disclosure of Invention
The purpose of the invention is as follows: the invention provides a preparation method of oligonucleotide (Oligo) carrier microspheres, which can effectively immobilize oligonucleotides. The microsphere can effectively immobilize oligonucleotide (Oligo) molecules, has good monodispersity and large oligonucleotide immobilization amount, and is favorable for capturing single-cell mRNA. The vector microsphere has good unicity, simple preparation method and large oligonucleotide immobilization capacity, and is beneficial to the application of single cell gene sequencing and the like.
The technical scheme is as follows: the invention aims to provide a preparation method of oligonucleotide (Oligo) carrier microspheres, which comprises the steps of mixing and reacting Oligo @ HB-PEGDA and molecules capable of reacting with HB-PEGDA to prepare water-in-oil droplets, and forming gel microspheres by polymerization and crosslinking by using the water-in-oil droplets as templates.
The preparation method comprises the following steps of (1) carrying out Michael addition reaction on the chemically modified Oligo fragment and hyperbranched PEGDA to prepare and obtain the difunctional HB-PEGDA; (2) taking the dual-functionalized HB-PEGDA as a basic unit, adding molecules capable of further reacting with the HB-PEGDA, and mixing to prepare a water-in-oil liquid drop; (3) and (3) initiating molecules in the liquid drop to polymerize under a proper condition to obtain gel microspheres, and washing out the gel microspheres to obtain the Oligo carrier microspheres.
In the preparation method, the chemically modified Oligo fragment in the step (1) is a thiol (-SH) group, an amino (-NH) group2) A modified Oligo fragment.
In the preparation method, the molecules capable of further reacting with HB-PEGDA in the step (2) are polyvinyl polyethylene glycol, sulfhydrylated functional polymers, double-bond functional polymers, amino functional polymers, polymeric monomers and the like.
In the preparation method, the polyvinyl polyethylene glycol can be multi-arm polyethylene glycol, tree-shaped polyethylene glycol and hyperbranched polyethylene glycol. The multi-Arm polyethylene glycol is respectively two-Arm polyethylene glycol ((Propargyl-PEG)2-Allyl), three-Arm polyethylene glycol (3Arm (PEG-Allyl3), four-Arm polyethylene glycol (4Arm (PEG-Allyl)4), six-Arm polyethylene glycol (6Arm-PEG-DA)), eight-Arm polyethylene glycol maleimide (8Arm-PEG-MAL), tree-shaped polyethylene glycol ((mPEG)4- (PEG)2-MAL), hyperbranched polyethylene glycol (hyperbranched polyethylene glycol diacrylate (HB-PEGDA)).
In the preparation method, the thiolated functional polymer can be thiolated hyaluronic acid and thiolated sodium alginate; the double-bond functional polymer can be double-bond gelatin, double-bond polyethylene glycol and the like.
The preparation method is characterized in that the polymerization monomer is one or more of acrylamide, dimethylacrylamide, isopropylacrylamide, hydroxyethyl methyl acrylate or polyethylene glycol acrylate.
In the preparation method, the water-in-oil droplet prepared in the step (2) is prepared by suspension polymerization, membrane emulsification and droplet microfluidics; the size range of the liquid drops is 1-1000 mu m.
The above production method is characterized in that the polymerization of the molecules inside the droplets is initiated under the appropriate conditions in the step (3) in such a manner that the molecules are mercapto (-SH), amino (-NH)2) The compound is subjected to Michael addition with the difunctional HB-PEGDA, and can also be prepared by using one or more of 2959, KPS or APS as a photoinitiator or a thermal initiator and then performing thermal polymerization or photoinitiated polymerization.
In the preparation method, in the step (3), the micron-sized hydrogel-in-oil microspheres are demulsified by adopting a sodium dodecyl sulfate solution, an alcohol, a salt solution or an ultrasonic method and then washed to obtain Oligo carrier microspheres.
Has the advantages that: compared with the prior art, the invention has the following remarkable effects: (1) the operation is simple, and the direct polymerization of the functional polymer microspheres is realized; (2) good controllability including regulation of microsphere reactive groups, regulation of microsphere size and regulation of the amount of the microspheres loaded with Oligo; (3) the maximum Oligo fragment loading capacity of the microspheres is large, single-cell gene sequencing is facilitated, the preparation method is simple, and the cost is low.
Drawings
FIG. 1 is a reaction scheme for the synthesis of HB-PEGDA;
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of a synthesized chemopolymer (HB-PEGDA);
FIG. 3 is a schematic diagram of a monodisperse Oligo carrier microsphere prepared by microfluidic technology.
Detailed Description
Example 1
(1) Synthesis of hyperbranched polyethylene glycol (HB-PEGDA) macromolecule: 11.5g of PEGDA monomer (average Mn. 575 g. mol.) was added-1) The resulting mixture was put into a flask, 50mL of butanone was added thereto, and the mixture was stirred until it was completely dissolved, thereby obtaining a monomer concentration of 0.4 mol. L-1The solution of (1). 0.1839g of Azobisisobutyronitrile (AIBN) and 0.2372g of tetraethylthiuram Disulfide (DS) were then added to the flask, the flask was sealed with a brine stopper and a sealing film was wound, and argon gas was bubbled through the mixture for 1 h. After preheating the oil bath to 70 ℃, the argon-filled mixture was placed at 70 ℃ for polymerization and the rotation speed was set at 300 rpm. After 6h of reaction, the reaction was stopped, air was bubbled through and the mixture was cooled to room temperature. The polymer was purified by precipitation three times with hexane/ether solution (1: 2v/v) (precooled in the refrigerator for 6h), the product was dried under reduced pressure and then dried in a vacuum oven for 48h to remove residual solvent to give the desired product.
(2) Preparation of thiolated hyaluronic acid: HA (100mg, 0.25mmol) was dissolved in 10ml MES buffer (pH 4.75, 0.1M), DTP (119mg, 0.5mmol) was added and dissolved with stirring; EDCI (96mg, 0.5mmol) was added, the pH of the solution was maintained at 4.75, and the reaction was carried out for 5 h; adding 1.0mol/L NaOH to adjust the pH of the reaction mixture to 7.0; DTT (501mg, 3.25mmol) was added and the pH of the solution was adjusted to 8.5 by the addition of 1.0 mol/LNaOH; the mixture was stirred for 24 hours and the pH of the reaction mixture was adjusted to 3.5 by addition of 1.0mol/L HCl; the acidified solution was transferred to a dialysis belt (molecular weight cut-off of 10kDa) and dialyzed against dilute HCl containing 0.1M NaCl for 24h (pH 3.5) followed by dialysis against deionized water for 24h (pH 3.5) with dialysis fluid changed every 12 h. The acidified solution was lyophilized for 72h to obtain solid HA-SH.
(3) Functional modification of HB-PEGDA with Oligo primers (Oligo @ HB-PEGDA): 75mg of HB-PEGDA was dissolved in 250. mu.L of PBS solution to give a 30% HB-PEGDA solution. 250 μ L of an oligo solution of a certain concentration was mixed with the prepared HB-PEGDA solution, and then the pH thereof was adjusted to weak alkalinity with a 1M NaOH solution, and the solution was incubated at 37 ℃ for 12 hours with shaking at 600 rpm. And (3) adjusting the pH value of the solution to be alkalescent by using a 1M NaOH solution to obtain an Oligo @ HB-PEGDA macromolecular solution with the Oligo fragments and double bonds being double-functionalized.
(4) Preparation of Oligo vector microspheres: HFE-7500 at 0.1% (v/v) surfactant was formulated as continuous phase P3, and 3mL of the solution was loaded into syringe A. Syringe B was filled with Oligo @ HB-PEGDA polymer solution. 2.9mg of tris (2-carboxyethyl) phosphonium (TECP) was weighed out and dissolved in 1mL of PBS to prepare a 10mM (TECP) solution. 20mg of thiolated hyaluronic acid (SH-HA) was weighed out and dissolved in 1mL of a (TECP) solution, and then the pH was adjusted to weak alkalinity with 1M NaOH solution, which was filled into two syringes C1 and C2. Each syringe contained 500 μ LSH-HA solution. Syringes of a, B, C1 and C2 were connected to the respective inlets of the microfluidic chip by PTFE tubes. Each syringe was housed in a syringe pump. For syringe A, the flow rate was set to 1000. mu.L/h, for syringe B of HBPEG solution, the flow rate was set to 100. mu.L/h, and for each of syringes C1 and C2 of oligo dT SH-HA solution, the droplet generation process was as shown in FIG. 3. And connecting the PTFE tube with an outlet of the microfluidic chip, collecting the micro-droplets by using a centrifugal tube after the droplet generation process is stable, standing for 2 hours after the collection is finished, removing the oil phase after the micro-droplets are solidified, and adding pure HFE-7500 to wash for multiple times to remove the surfactant. HFE-7500 was removed and the resulting oligo carrier hydrogel microspheres were dispersed in PBS.
Example 2
(1) Synthesis of hyperbranched polyethylene glycol (HB-PEGDA) macromolecule: 11.5g of PEGDA monomer (average Mn. 575 g. mol.) was added-1) The resulting mixture was put into a flask, 50mL of butanone was added thereto, and the mixture was stirred until it was completely dissolved, thereby obtaining a monomer concentration of 0.4 mol. L-1The solution of (1). 0.1839g of Azobisisobutyronitrile (AIBN) and 0.2372g of tetraethylthiuram Disulfide (DS) were then added to the flask, the flask was sealed with a stopper of saline and a sealing film was wound and argon was bubbled through the mixture for 1 h. After preheating the oil bath to 70 ℃, the argon-filled mixture was placed at 70 ℃ for polymerization and the rotation speed was set at 300 rpm. After 6h of reaction the reaction is stoppedAir was then introduced and the mixture was cooled to room temperature. The polymer was purified by precipitation three times with hexane/ether solution (1: 2v/v) (precooled in the refrigerator for 6h), the product was dried under reduced pressure and then dried in a vacuum oven for 48h to remove residual solvent to give the desired product.
(2) Preparation of thiolated hyaluronic acid: HA (100mg, 0.25mmol) was dissolved in 10ml MES buffer (pH 4.75, 0.1M), DTP (119mg, 0.5mmol) was added and dissolved with stirring; EDCI (96mg, 0.5mmol) was added, the pH of the solution was maintained at 4.75, and the reaction was carried out for 5 h; adding 1.0mol/L NaOH to adjust the pH of the reaction mixture to 7.0; DTT (501mg, 3.25mmol) was added and the pH of the solution was adjusted to 8.5 by the addition of 1.0 mol/LNaOH; the mixture was stirred for 24 hours and the pH of the reaction mixture was adjusted to 3.5 by addition of 1.0mol/L HCl; the acidified solution was transferred to a dialysis belt (molecular weight cut-off of 10kDa) and dialyzed against dilute HCl containing 0.1M NaCl for 24h (pH 3.5) followed by dialysis against deionized water for 24h (pH 3.5) with dialysis fluid changed every 12 h. The acidified solution was lyophilized for 72h to obtain solid HA-SH.
(3) Functional modification of HB-PEGDA with Oligo primers (Oligo @ HB-PEGDA): 75mg of HB-PEGDA was dissolved in 250. mu.L of PBS solution to give a 30% HB-PEGDA solution. 250 μ L of an oligo solution of a certain concentration was mixed with the prepared HB-PEGDA solution, and then the pH thereof was adjusted to weak alkalinity with a 1M NaOH solution, and the solution was incubated at 37 ℃ for 12 hours with shaking at 600 rpm. And (3) adjusting the pH value of the solution to be alkalescent by using a 1M NaOH solution to obtain an Oligo @ HB-PEGDA macromolecular solution with the Oligo fragments and double bonds being double-functionalized.
(4) Preparation of Oligo vector microspheres: methyl silicone oil with 5% (v/v) surfactant (749) was formulated as continuous phase P3, and 3mL of the solution was loaded into syringe a. Syringe B was filled with Oligo @ HB-PEGDA polymer solution. 2.9mg of tris (2-carboxyethyl) phosphonium (TECP) was weighed out and dissolved in 1mL of PBS to prepare a 10mM (TECP) solution. 20mg of thiolated hyaluronic acid (SH-HA) was weighed out and dissolved in 1mL of a (TECP) solution, and then the pH was adjusted to weak alkalinity with 1M NaOH solution, which was filled into two syringes C1 and C2. Each syringe contained 500. mu.L of SH-HA solution. Syringes of a, B, C1 and C2 were connected to the respective inlets of the microfluidic chip by PTFE tubes. Each syringe was housed in a syringe pump. For syringe A, the flow rate was set to 1000. mu.L/h, for syringe B of HBPEG solution, the flow rate was set to 100. mu.L/h, and for each of syringes C1 and C2 of oligo dT SH-HA solution, the droplet generation process was as shown in FIG. 3. And connecting the PTFE tube with an outlet of the microfluidic chip, collecting the micro-droplets by using a centrifugal tube after the droplet generation process is stable, standing for 2 hours after the collection is finished, removing the oil phase after the micro-droplets are solidified, and adding n-hexane for washing for multiple times to remove the surfactant. The n-hexane was removed and the oligo carrier microspheres were dispersed in PBS.

Claims (9)

1. A preparation method of oligonucleotide (Oligo) carrier microspheres is characterized in that: mixing Oligo @ HB-PEGDA and molecules capable of reacting with HB-PEGDA to prepare a water-in-oil droplet, and using the water-in-oil droplet as a template to form gel microspheres through polymerization and crosslinking;
the preparation method comprises the following steps:
(1) carrying out Michael addition reaction on the chemically modified Oligo fragment and HB-PEGDA to prepare Oligo @ HB-PEGDA;
(2) taking Oligo @ HB-PEGDA as a basic unit, adding molecules which can further react with the HB-PEGDA, and mixing to prepare a water-in-oil droplet;
(3) and (3) initiating molecules inside the liquid drops to polymerize under a proper condition to obtain gel microspheres, and purifying the gel microspheres to obtain the Oligo carrier microspheres.
2. The method of claim 1, wherein the oligonucleotide (Oligo) vector microsphere is prepared by: the Oligo fragment chemically modified in the step (1) is a thiol (-SH) group or an amino (-NH) group2) The modified Oligo fragment has a sequence of a macromolecular polymer consisting of deoxynucleotides, wherein bases comprise one or more of adenine (A), guanine (G), thymine (T) and cytosine (C), the bases are arranged in any sequence, and the number of the bases is 1-50.
3. The method of claim 1, wherein the oligonucleotide (Oligo) vector microsphere is prepared by: the molecules which can further react with HB-PEGDA in the step (2) are polyvinyl polyethylene glycol, sulfhydrylation functional polymers, double bond functional polymers, amino functional polymers, small molecular monomers and the like.
4. The method for preparing oligonucleotide (Oligo) vector microspheres according to claim 3, wherein: the polyvinyl polyethylene glycol can be multi-arm polyethylene glycol, tree polyethylene glycol and hyperbranched polyethylene glycol. The multi-Arm polyethylene glycol is respectively two-Arm polyethylene glycol ((Propargyl-PEG)2-Allyl), three-Arm polyethylene glycol (3Arm (PEG-Allyl3), four-Arm polyethylene glycol (4Arm (PEG-Allyl)4), six-Arm polyethylene glycol (6Arm-PEG-DA)), eight-Arm polyethylene glycol maleimide (8Arm-PEG-MAL), tree-shaped polyethylene glycol ((mPEG)4- (PEG)2-MAL), hyperbranched polyethylene glycol (hyperbranched polyethylene glycol diacrylate (HB-PEGDA)).
5. The method for preparing oligonucleotide (Oligo) vector microspheres according to claim 3, wherein: the sulfhydrylation functional polymer can be one or more of sulfhydrylation hyaluronic acid, sulfhydrylation sodium alginate, sulfhydrylation gelatin and the like; the double-bond functional polymer can be one or more of double-bond gelatin, double-bond polyethylene glycol and the like.
6. The method for preparing oligonucleotide (Oligo) vector microspheres according to claim 3, wherein: the polymerization monomer is one or more of acrylamide, dimethylacrylamide, isopropyl acrylamide, hydroxyethyl methyl acrylate or polyethylene glycol acrylate.
7. The method for preparing oligonucleotide (Oligo) carrier microspheres according to claim 1, wherein: the water-in-oil droplet prepared in the step (2) is prepared by suspension polymerization, membrane emulsification and droplet microfluidics; the size of the droplets is in the range of 1 μm to 1000. mu.m.
8. The method of claim 1, wherein the oligonucleotide (Oligo) vector microsphere is prepared by: the polymerization of the molecules in the liquid drops under the proper conditions in the step (3) can be initiated by sulfydryl (-SH) and amino (-NH)2) The compound is subjected to Michael addition with the difunctional HB-PEGDA, and can also be prepared by using one or more of 2959, KPS or APS as a photoinitiator or a thermal initiator and then performing thermal polymerization or photoinitiated polymerization.
9. The method of claim 1, wherein the oligonucleotide (Oligo) vector microsphere is prepared by: and (3) demulsifying the micron-sized hydrogel-in-oil microspheres by adopting a sodium dodecyl sulfate solution, an alcohol, a salt solution or an ultrasonic method, and then washing to obtain Oligo carrier microspheres.
CN202110445594.7A 2021-04-23 2021-04-23 Preparation method of oligonucleotide carrier microsphere Active CN113058511B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110445594.7A CN113058511B (en) 2021-04-23 2021-04-23 Preparation method of oligonucleotide carrier microsphere

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110445594.7A CN113058511B (en) 2021-04-23 2021-04-23 Preparation method of oligonucleotide carrier microsphere

Publications (2)

Publication Number Publication Date
CN113058511A CN113058511A (en) 2021-07-02
CN113058511B true CN113058511B (en) 2022-05-13

Family

ID=76567509

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110445594.7A Active CN113058511B (en) 2021-04-23 2021-04-23 Preparation method of oligonucleotide carrier microsphere

Country Status (1)

Country Link
CN (1) CN113058511B (en)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112458075B (en) * 2020-11-24 2023-07-25 南京工业大学 Method for strengthening microbial catalytic process by using double cross-linked particle gel packed column
CN113429523B (en) * 2021-07-09 2023-05-09 西南石油大学 Core-shell polymer microsphere and preparation method thereof
CN113797904A (en) * 2021-09-30 2021-12-17 苏州赛分科技股份有限公司 Chromatographic medium for capturing mRNA and preparation method thereof
CN114773608A (en) * 2022-04-18 2022-07-22 南京工业大学 Long-acting hyaluronic acid for treating osteoarthritis and preparation method thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112048033B (en) * 2020-09-04 2021-10-29 湖南大学 Hydrogel microcarrier and preparation method and application thereof

Also Published As

Publication number Publication date
CN113058511A (en) 2021-07-02

Similar Documents

Publication Publication Date Title
CN113058511B (en) Preparation method of oligonucleotide carrier microsphere
CN105925572B (en) DNA coding microsphere and synthetic method thereof
AU2019346419B2 (en) Three-dimensional bioreactors
JP2020509746A (en) Microgel particles and related compositions, systems, and methods for use in 3D printing and 3D cell culture media
CN101633712A (en) Point-and-click temperature/pH-sensibility microgel
CN101235117A (en) Copolymerization reaction method for styrene/maleic anhydride
CN109400888B (en) Water-soluble star polymer based on beta-cyclodextrin as core and preparation method thereof
Lee et al. The mechanism of core–shell inversion in two‐stage latexes
CN111195371A (en) Micro-heterotype cell-carrying alginic acid gel and preparation method and application thereof
CN101381435B (en) Preparation method of spherical polyelectrolyte brush and use thereof
CN104262554B (en) Self-crosslinkable responsive to temperature type hybrid block copolymer and preparation method thereof
CN104744650A (en) Preparation and application of hybrid copolymer containing POSS (polyhedral oligomeric silsesquioxane) base
CN104387712A (en) Nano composite carrier with superparamagnetism and preparation method thereof
CN110585476B (en) Preparation method of non-spherical hydrogel microparticle embolic agent
CN1278534A (en) Polymer microball with fluorescent mark and its preparation
CN103130951A (en) Method for preparing P(SMA-co-DM) in micro-reactor through free radical polymerization
CN103865012A (en) Preparation of polymer-polypeptide bioconjugate with comb-shaped structure
CN107236078B (en) Preparation method of flexible soft monomer colloid photonic crystal emulsion
CN111269368B (en) Method for preparing surface microphase separation nanoparticles by RAFT polymerization induced self-assembly
CN103394305A (en) pH-responsive star-shaped polymer emulsifier and preparation method thereof
CN115505160B (en) Preparation method of hydrogel microsphere carrier, product obtained by preparation method and application of hydrogel microsphere carrier
CN109897179B (en) Multi-arm polyethylene glycol-poly (L-glutamate) block copolymer, and preparation method and application thereof
CN115536790A (en) Star polymer/metal nanoparticle compound with stimulus response function and continuous flow microreaction preparation method and application thereof
Huang et al. Synthesis and DNA adsorption of poly (2-Vinyl-4, 6-Diamino-1, 3, 5-Triazine) coated polystyrene microspheres
Daniels et al. Overview of Polymer Colloids: Preparation, Characterization, and Applications

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant