WO2022236863A1 - Microsphère ayant un point fonctionnalisé à sa surface, procédé de préparation et application de la microsphère - Google Patents

Microsphère ayant un point fonctionnalisé à sa surface, procédé de préparation et application de la microsphère Download PDF

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Publication number
WO2022236863A1
WO2022236863A1 PCT/CN2021/095043 CN2021095043W WO2022236863A1 WO 2022236863 A1 WO2022236863 A1 WO 2022236863A1 CN 2021095043 W CN2021095043 W CN 2021095043W WO 2022236863 A1 WO2022236863 A1 WO 2022236863A1
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microsphere
functionalized
functionalized spots
spots
surface according
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PCT/CN2021/095043
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English (en)
Chinese (zh)
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徐方成
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厦门依加成科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/10—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
    • B01J20/103—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate comprising silica
    • 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
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/261—Synthetic macromolecular compounds obtained by reactions only involving carbon to carbon unsaturated bonds
    • 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
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
    • B01J20/28004—Sorbent size or size distribution, e.g. particle size
    • B01J20/28007—Sorbent size or size distribution, e.g. particle size with size in the range 1-100 nanometers, e.g. nanosized particles, nanofibers, nanotubes, nanowires or the like
    • 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
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
    • B01J20/28009—Magnetic properties
    • 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
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B01J20/28016—Particle form
    • B01J20/28021—Hollow particles, e.g. hollow spheres, microspheres or cenospheres
    • 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
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B01J20/28047—Gels
    • 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
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28054—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their surface properties or porosity
    • B01J20/28078—Pore diameter
    • B01J20/28085—Pore diameter being more than 50 nm, i.e. macropores
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/02—Separating microorganisms from their culture media
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Definitions

  • the invention relates to the technical field of biomaterials, in particular to a microsphere with functionalized spots on the surface and a preparation method and application thereof.
  • Cells, bacteria, and viruses are the three major categories of materials in the field of biology. They all have the properties of particles. For example, viruses are usually 15-200nm, bacteria are 0.2-8 ⁇ m, and human mammalian cells are usually 5-30 ⁇ m.
  • Existing biological research methods whether it is physiological and biochemical research, or gene and genome research, usually use many of the same cells. For example, in conventional bacterial genome sequencing, hundreds of thousands or even millions of bacterial individuals are usually used to crush together and extract genes as templates, and then perform gene amplification and sequencing.
  • modern science and technology have proved that there are differences between individuals of bacteria, viruses, and cells.
  • the individual viruses are extremely small and belong to nanoscale biological particles, they can usually only be seen under an electron microscope. It is still difficult to separate a large number of individual virus particles with high throughput. Therefore, it is necessary to find new materials or methods that can be used for the isolation of virus single particles, so as to be applied to the isolation and purification of viruses.
  • the existing technology usually uses methods such as flow cytometry and micro-manipulation under an optical microscope to divide bacteria or cells into several categories.
  • the single-cell instrument of 10xGenomics uses microfluidics. Isolate single cells, but usually only cells larger than 5 ⁇ m can be separated, and the highest yield is only about 40%, usually only a dozen percent. Therefore, it is also necessary to develop single-cell isolation methods and instruments based on new principles.
  • the present invention provides a microsphere with functionalized micro or nano spots on the surface, its preparation method and application.
  • the present invention adopts following technical scheme:
  • a microsphere with functionalized spots on the surface comprising a microsphere body and a functionalized spot inlaid on the top of the microsphere body, a gravity ball is embedded in the bottom of the microsphere body, and the gravity ball is used to adjust the The center of gravity of the microsphere body keeps the functionalized spots upward.
  • the diameter of the microsphere body is 2-60 ⁇ m
  • the surface does not have adsorption properties
  • the surface is modified with fluorescence or quantum dots
  • the inside is magnetically modified.
  • microsphere main body adopts polystyrene magnetic microspheres or silica magnetic microspheres.
  • the diameter of the gravity ball is 30%-50% of the main body of the microsphere, and the specific gravity is 2-10.
  • the functionalized spots have adsorption properties and have a diameter of 15nm-30 ⁇ m.
  • the functionalized spots contain substances that can adsorb bioactive particles
  • the substances that can adsorb bioactive particles include adsorbed molecules, adsorbed nanoparticles or modified micro-nanospheres.
  • the functionalized spot is embedded on the top of the microsphere body through gel fixation.
  • the gel is gelatin, agar or polyethylene glycol.
  • the adsorption molecules include streptavidin, protein A or protein G;
  • the adsorption nanoparticles include gold nanoparticles and fullerene nanoparticles with a diameter of 1-20nm;
  • the modified micro-nanospheres It is a micro-nanosphere modified by amino group, carboxyl group, hydroxyl group or streptavidin, with a diameter of 2nm-30 ⁇ m.
  • a method for preparing microspheres with functionalized spots on the surface comprising the steps of:
  • the diameter of the micron hole in step S1 is 2-60 ⁇ m, and the depth is 1-30 ⁇ m.
  • the thickness of the nano-film in step S4 is 3-50 nm; the diameter of the micro-nanopore is 15 nm-30 ⁇ m, and the depth is 15 nm-30 ⁇ m.
  • microspheres with functionalized spots on the surface for the adsorption and separation of biologically active particles.
  • the biologically active particles include virus single particles, protein single particles, nucleic acid single particles, single microorganisms or single cells.
  • the adsorption and separation of the biologically active particles specifically includes the following steps:
  • the present invention has the following advantages compared with the background technology:
  • the bottom of the microsphere of the present invention is embedded with a gravity ball, and the center of gravity of the microsphere body is adjusted through the gravity ball, so that the microsphere body is in a tumbler state in the solution, so that the functionalized spot on the top of the microsphere body can always keep upward , which is conducive to the adsorption, detection and separation of bioactive particles;
  • the functionalized spots on the top of the microspheres contain active substances that adsorb biologically active particles, so they have a good ability to adsorb biologically active particles. According to the size of the biologically active particles, functionalized spots with nanometer or micron size similar to the size of the active particles can be selected.
  • nano-functionalized spots similar in size to virus particles can be designed, and single virus particles are adsorbed on the functionalized spots, while the main surface of the microsphere does not adsorb Biological particles, the surface of which is modified by fluorescence or quantum dots, and the interior is magnetically modified, and the separation and detection of microspheres can be realized by magnetic adsorption method;
  • the preparation method of the microspheres of the present invention has the advantages of low cost, high efficiency and rapidity
  • microspheres of the present invention have the functions of adsorption and separation of bioactive particles.
  • Fig. 1 is the schematic diagram of microsphere structure and application principle of the present invention
  • Fig. 2 is a schematic diagram of functionalized spots prepared by imprinting in the present invention.
  • a microsphere with functionalized spots on the surface includes a microsphere main body 1 and a functionalized spot 2 embedded on the top of the microsphere main body 1, and the bottom of the microsphere main body 1 is embedded with gravity Ball 3, the gravity ball 3 is used to adjust the center of gravity of the microsphere body 1, so that the functionalized spot 2 keeps upward.
  • the functionalized spots 2 are used to adsorb individual bioactive particles 4 .
  • the microsphere main body 1 adopts commercially available polystyrene magnetic microspheres with a diameter of 5 ⁇ m.
  • the gravity ball 3 has a diameter of 2.5 ⁇ m and a specific gravity of 7.6.
  • the functionalized spot 2 is solidified from a mixed gel containing streptavidin, has adsorption properties, and has a diameter of 100 nm.
  • the mixed gel is composed of 10% gelatin and 0.15% streptavidin.
  • gelatin with a concentration of 8-12% and a mass concentration of 0.05%-0.3% streptavidin can be selected for the gel.
  • a kind of surface has the preparation method of the microsphere of functionalized spot, comprises the steps:
  • the diameter of the cylindrical hole can be selected to be 50-400nm larger than the diameter of the microsphere, the hole depth is 30-750nm smaller than the diameter of the microsphere, the distance between the walls of two adjacent holes is 3-25 ⁇ m, and the thickness of the porous ceramic chip
  • the chip 5 can also choose a single crystal silicon plate chip with a thickness of 2-20 ⁇ m, and the holes of the micron hole array can also be made into tapered holes.
  • the microsphere body can be controlled to be 30-750nm higher than the surface of the chip 5; in addition, the above steps can also adopt the method of filtering to array the microsphere body 1 on the chip 5.
  • the thickness of the optional nano film 7 is 5-50nm
  • the gel of this embodiment uses The commercially available gelatin has a concentration of 10%, and the adsorbent is commercially available surface aminated silica nanospheres with a diameter of 10 nm.
  • the gel can also be made of polymer materials such as agar and polyethylene glycol.
  • microspheres of Example 1 are used for adsorption and separation of virus single particles, which specifically includes the following steps:
  • A2 adding a solution containing virus particles to the surface of the first chip, and the virus particles are adsorbed on the functionalized spots of the microspheres;
  • microspheres with functionalized spots of corresponding size are designed, so that each functionalized spot can only adsorb a single virus particle, thereby realizing the separation of single virus particles.
  • corresponding functionalized spots can be designed according to the size of the bacteria and cells that need to be adsorbed and separated, so as to realize the single particle separation of bioactive particles of different sizes.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Nanotechnology (AREA)
  • Molecular Biology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Biophysics (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Inorganic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Dispersion Chemistry (AREA)
  • Cell Biology (AREA)
  • Immunology (AREA)
  • Materials Engineering (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Une microsphère ayant un point fonctionnalisé à sa surface, comprenant un corps principal de microsphère et un point fonctionnalisé incorporé dans la partie supérieure du corps principal de microsphère. Une bille à gravité est incorporée dans le fond du corps principal de microsphère, et la bille de gravité est utilisée pour ajuster le centre de gravité du corps principal de microsphère pour maintenir le point fonctionnalisé vers le haut. L'invention concerne en outre un procédé de préparation et une application de la microsphère. La microsphère ayant le point fonctionnalisé peut être utilisée pour l'adsorption et la séparation de particules bioactives.
PCT/CN2021/095043 2021-05-14 2021-05-21 Microsphère ayant un point fonctionnalisé à sa surface, procédé de préparation et application de la microsphère WO2022236863A1 (fr)

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CN202110528778.X 2021-05-14
CN202110528778.XA CN113265379B (zh) 2021-05-14 2021-05-14 一种表面具有功能化斑点的微球及其制备方法和应用

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Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114307885B (zh) * 2022-01-20 2022-09-16 厦门大学 一种局部功能化修饰微球的制备方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5945525A (en) * 1995-07-07 1999-08-31 Toyo Boseki Kabushiki Kaisha Method for isolating nucleic acids using silica-coated magnetic particles
US20070117089A1 (en) * 2005-11-21 2007-05-24 Croker Kevin M Sol-gel coated glass microspheres for use in bioassay
WO2007140497A1 (fr) * 2006-06-02 2007-12-13 Universität Linz Nanoréseau de virus
CN111393574A (zh) * 2020-03-31 2020-07-10 中国科学院过程工程研究所 一种表面带有功能基团的磁性微球及其制备方法和用途
CN112111042A (zh) * 2019-06-21 2020-12-22 康码（上海）生物科技有限公司 一种生物磁性微球及其制备方法和使用方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5945525A (en) * 1995-07-07 1999-08-31 Toyo Boseki Kabushiki Kaisha Method for isolating nucleic acids using silica-coated magnetic particles
US20070117089A1 (en) * 2005-11-21 2007-05-24 Croker Kevin M Sol-gel coated glass microspheres for use in bioassay
WO2007140497A1 (fr) * 2006-06-02 2007-12-13 Universität Linz Nanoréseau de virus
CN112111042A (zh) * 2019-06-21 2020-12-22 康码（上海）生物科技有限公司 一种生物磁性微球及其制备方法和使用方法
CN111393574A (zh) * 2020-03-31 2020-07-10 中国科学院过程工程研究所 一种表面带有功能基团的磁性微球及其制备方法和用途

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CN113265379B (zh) 2022-04-29

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