CN113058511A - Preparation method of oligonucleotide carrier microsphere - Google Patents

Preparation method of oligonucleotide carrier microsphere Download PDF

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CN113058511A
CN113058511A CN202110445594.7A CN202110445594A CN113058511A CN 113058511 A CN113058511 A CN 113058511A CN 202110445594 A CN202110445594 A CN 202110445594A CN 113058511 A CN113058511 A CN 113058511A
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polyethylene glycol
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余子夷
文慧琳
张静
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Nanjing Tech University
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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 μm.
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 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 polymerized at 70 ℃ and the rotational speed was setAt 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 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: 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 (10)

1. A method for preparing oligonucleotide (Oligo) carrier microspheres is characterized in that: the gel microsphere is prepared by mixing Oligo @ HB-PEGDA and molecules capable of reacting with HB-PEGDA to form water-in-oil droplets, and using the water-in-oil droplets as a template and performing polymerization and crosslinking.
2. The method of claim 1, wherein the oligonucleotide (Oligo) vector microsphere is prepared by: the 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) adding molecules which can further react with HB-PEGDA into Oligo @ HB-PEGDA serving as a basic unit, 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.
3. The method for preparing oligonucleotide (Oligo) vector microspheres according to claim 2, wherein: 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.
4. The method for preparing oligonucleotide (Oligo) vector microspheres according to claim 2, wherein: 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.
5. The method of claim 4, wherein the oligonucleotide (Oligo) vector microsphere is prepared by: 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)).
6. The method of claim 4, wherein the oligonucleotide (Oligo) vector microsphere is prepared by: 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.
7. The method of claim 4, wherein the oligonucleotide (Oligo) vector microsphere is prepared by: the polymerization monomer is one or more of acrylamide, dimethylacrylamide, isopropyl acrylamide, hydroxyethyl methyl acrylate or polyethylene glycol acrylate.
8. The method for preparing oligonucleotide (Oligo) vector microspheres according to claim 2, wherein: the water-in-oil droplets prepared in the step (2) are prepared by suspension polymerization, membrane emulsification and droplet microfluidics; the droplets have a particle size in the range of 1 μm to 1000 μm.
9. The method for preparing oligonucleotide (Oligo) vector microspheres according to claim 2, wherein: 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.
10. The method for preparing oligonucleotide (Oligo) vector microspheres according to claim 2, wherein: 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.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112458075A (en) * 2020-11-24 2021-03-09 南京工业大学 Method for reinforcing microbial catalysis process by using double-crosslinked-particle gel packed column
CN113429523A (en) * 2021-07-09 2021-09-24 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

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112048033A (en) * 2020-09-04 2020-12-08 湖南大学 Hydrogel microcarrier and preparation method and application thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112048033A (en) * 2020-09-04 2020-12-08 湖南大学 Hydrogel microcarrier and preparation method and application thereof

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112458075A (en) * 2020-11-24 2021-03-09 南京工业大学 Method for reinforcing microbial catalysis process by using double-crosslinked-particle gel packed column
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
CN113429523A (en) * 2021-07-09 2021-09-24 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

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